Elucidating Functional Aspects of P-type ATPases

DEFF Research Database (Denmark)

Autzen, Henriette Elisabeth

2015-01-01

and helped enlighten how thapsigargin, a potent inhibitor of SERCA1a, depends on a water mediated hydrogen bond network when bound to SERCA1a. Furthermore, molecular dynamics (MD) simulations of the same P-type ATPase were used to assess a long-standing question whether cholesterol affects SERCA1a through...... similar to that of the wild type (WT) protein. The discrepancy between the newly determined crystal structure of LpCopA and the functional manifestations of the missense mutation in human CopA, could indicate that LpCopA is insufficient in structurally elucidating the effect of disease-causing mutations...... in the human CopA proteins. MD simulations, which combine coarse-grained (CG) and atomistic procedures, were set up in order to elucidate mechanistic implications exerted by the lipid bilayer on LpCopA. The MD simulations of LpCopA corroborated previous and new in vivo activity data and showed...

Expression of a prokaryotic P-type ATPase in E. coli Plasma Membranes and Purification by Ni2+-affinity chromatography

Directory of Open Access Journals (Sweden)

Geisler Markus

1998-01-01

Full Text Available In order to characterize the P-type ATPase from Synechocystis 6803 [Geisler (1993 et al. J. Mol. Biol. 234, 1284] and to facilitate its purification, we expressed an N-terminal 6xHis-tagged version of the ATPase in an ATPase deficient E. coli strain. The expressed ATPase was immunodetected as a dominant band of about 97 kDa localized to the E. coli plasma membranes representing about 20-25% of the membrane protein. The purification of the Synecho-cystis 6xHis-ATPase by single-step Ni-affinity chromatography under native and denaturating conditions is described. ATPase activity and the formation of phosphointermediates verify the full function of the enzyme: the ATPase is inhibited by vanadate (IC50= 119 &mgr;M and the formation of phosphorylated enzyme intermediates shown by acidic PAGE depends on calcium, indicating that the Synechocystis P-ATPase functions as a calcium pump.

Tetrahydrocarbazoles are a novel class of potent P-type ATPase inhibitors with antifungal activity

DEFF Research Database (Denmark)

Bublitz, Maike; Kjellerup, Lasse; Cohrt, Karen O.Hanlon

2018-01-01

We have identified a series of tetrahydrocarbazoles as novel P-type ATPase inhibitors. Using a set of rationally designed analogues, we have analyzed their structure-activity relationship using functional assays, crystallographic data and computational modeling. We found that tetrahydrocarbazoles...

Ankyrin-B coordinates the Na/K ATPase, Na/Ca exchanger, and InsP3 receptor in a cardiac T-tubule/SR microdomain.

Directory of Open Access Journals (Sweden)

2005-12-01

Full Text Available We report identification of an ankyrin-B-based macromolecular complex of Na/K ATPase (alpha 1 and alpha 2 isoforms, Na/Ca exchanger 1, and InsP3 receptor that is localized in cardiomyocyte T-tubules in discrete microdomains distinct from classic dihydropyridine receptor/ryanodine receptor "dyads." E1425G mutation of ankyrin-B, which causes human cardiac arrhythmia, also blocks binding of ankyrin-B to all three components of the complex. The ankyrin-B complex is markedly reduced in adult ankyrin-B(+/- cardiomyocytes, which may explain elevated [Ca2+]i transients in these cells. Thus, loss of the ankyrin-B complex provides a molecular basis for cardiac arrhythmia in humans and mice. T-tubule-associated ankyrin-B, Na/Ca exchanger, and Na/K ATPase are not present in skeletal muscle, where ankyrin-B is expressed at 10-fold lower levels than in heart. Ankyrin-B also is not abundantly expressed in smooth muscle. We propose that the ankyrin-B-based complex is a specialized adaptation of cardiomyocytes with a role for cytosolic Ca2+ modulation.

Evolution of the P-type II ATPase gene family in the fungi and presence of structural genomic changes among isolates of Glomus intraradices

Directory of Open Access Journals (Sweden)

Sanders Ian R

2006-03-01

Full Text Available Abstract Background The P-type II ATPase gene family encodes proteins with an important role in adaptation of the cell to variation in external K+, Ca2+ and Na2+ concentrations. The presence of P-type II gene subfamilies that are specific for certain kingdoms has been reported but was sometimes contradicted by discovery of previously unknown homologous sequences in newly sequenced genomes. Members of this gene family have been sampled in all of the fungal phyla except the arbuscular mycorrhizal fungi (AMF; phylum Glomeromycota, which are known to play a key-role in terrestrial ecosystems and to be genetically highly variable within populations. Here we used highly degenerate primers on AMF genomic DNA to increase the sampling of fungal P-Type II ATPases and to test previous predictions about their evolution. In parallel, homologous sequences of the P-type II ATPases have been used to determine the nature and amount of polymorphism that is present at these loci among isolates of Glomus intraradices harvested from the same field. Results In this study, four P-type II ATPase sub-families have been isolated from three AMF species. We show that, contrary to previous predictions, P-type IIC ATPases are present in all basal fungal taxa. Additionally, P-Type IIE ATPases should no longer be considered as exclusive to the Ascomycota and the Basidiomycota, since we also demonstrate their presence in the Zygomycota. Finally, a comparison of homologous sequences encoding P-type IID ATPases showed unexpectedly that indel mutations among coding regions, as well as specific gene duplications occur among AMF individuals within the same field. Conclusion On the basis of these results we suggest that the diversification of P-Type IIC and E ATPases followed the diversification of the extant fungal phyla with independent events of gene gains and losses. Consistent with recent findings on the human genome, but at a much smaller geographic scale, we provided evidence

Towards the structure of yeast and mammalian P4-ATPases

DEFF Research Database (Denmark)

Lyons, Joseph; Laban, Milena; Mikkelsen, Stine

2017-01-01

P4-ATPases are members of the P-type ATPase superfamily that drive the inward translocation (flipping) of lipids within the membrane. These lipid flippase largely function as binary complexes with an auxiliary protein from the CDC50 family. The bulk of our knowledge has derived genetic and bioche......P4-ATPases are members of the P-type ATPase superfamily that drive the inward translocation (flipping) of lipids within the membrane. These lipid flippase largely function as binary complexes with an auxiliary protein from the CDC50 family. The bulk of our knowledge has derived genetic...... a basis for the analysis of reported mutagenesis data, we aim to solve the first molecular structures of the PS transporting P4-ATPases using electron microscopy. To date, negative stain EM analysis, on detergent, amphipol and saposin-lipoprotein nanoparticle (Salipro) reconstituted of both Drs2p/CDC50p...... and bATP8A2/CDC50A, has yielded comparable low-resolution envelopes of these two transporters, highlighting the bulk architecture of the complex. Current efforts and progress on the functional characterization and cryo-EM analysis of both lipid transporters reconstituted in Salipro are described...

A Non-Competitive Inhibitor of VCP/p97 and VPS4 Reveals Conserved Allosteric Circuits in Type I and II AAA ATPases.

Science.gov (United States)

Pöhler, Robert; Krahn, Jan H; van den Boom, Johannes; Dobrynin, Grzegorz; Kaschani, Farnusch; Eggenweiler, Hans-Michael; Zenke, Frank T; Kaiser, Markus; Meyer, Hemmo

2018-02-05

AAA ATPases have pivotal functions in diverse cellular processes essential for survival and proliferation. Revealing strategies for chemical inhibition of this class of enzymes is therefore of great interest for the development of novel chemotherapies or chemical tools. Here, we characterize the compound MSC1094308 as a reversible, allosteric inhibitor of the type II AAA ATPase human ubiquitin-directed unfoldase (VCP)/p97 and the type I AAA ATPase VPS4B. Subsequent proteomic, genetic and biochemical studies indicate that MSC1094308 binds to a previously characterized drugable hotspot of p97, thereby inhibiting the D2 ATPase activity. Our results furthermore indicate that a similar allosteric site exists in VPS4B, suggesting conserved allosteric circuits and drugable sites in both type I and II AAA ATPases. Our results may thus guide future chemical tool and drug discovery efforts for the biomedically relevant AAA ATPases. © 2018 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.

Phylogenetic analysis of P5 P-type ATPases, a eukaryotic lineage of secretory pathway pumps

DEFF Research Database (Denmark)

Møller, Annette; Asp, Torben; Holm, Preben Bach

2008-01-01

prokaryotic genome. Based on a protein alignment we could group the P5 ATPases into two subfamilies, P5A and P5B that, based on the number of negative charges in conserved trans-membrane segment 4, are likely to have different ion specificities. P5A ATPases are present in all eukaryotic genomes sequenced so......Eukaryotes encompass a remarkable variety of organisms and unresolved lineages. Different phylogenetic analyses have lead to conflicting conclusions as to the origin and associations between lineages and species. In this work, we investigated evolutionary relationship of a family of cation pumps...... exclusive for the secretory pathway of eukaryotes by combining the identification of lineage-specific genes with phylogenetic evolution of common genes. Sequences of P5 ATPases, which are regarded to be cation pumps in the endoplasmic reticulum (ER), were identified in all eukaryotic lineages but not in any...

Membrane Targeting of P-type ATPases in Plant Cells

International Nuclear Information System (INIS)

Harper, Jeffrey F.

2004-01-01

How membrane proteins are targeted to specific subcellular locations is a very complex and poorly understood area of research. Our long-term goal is to use P-type ATPases (ion pumps), in a model plant system Arabidopsis, as a paradigm to understand how members of a family of closely related membrane proteins can be targeted to different subcellular locations. The research is divided into two specific aims. The first aim is focused on determining the targeting destination of all 10 ACA-type calcium pumps (Arabidopsis Calcium ATPase) in Arabidopsis. ACAs represent a plant specific-subfamily of plasma membrane-type calcium pumps. In contrast to animals, the plant homologs have been found in multiple membrane systems, including the ER (ACA2), tonoplast (ACA4) and plasma membrane (ACA8). Their high degree of similarity provides a unique opportunity to use a comparative approach to delineate the membrane specific targeting information for each pump. One hypothesis to be tested is that an endomembrane located ACA can be re-directed to the plasma membrane by including targeting information from a plasma membrane isoform, ACA8. Our approach is to engineer domain swaps between pumps and monitor the targeting of chimeric proteins in plant cells using a Green Fluorescence Protein (GFP) as a tag. The second aim is to test the hypothesis that heterologous transporters can be engineered into plants and targeted to the plasma membrane by fusing them to a plasma membrane proton pump. As a test case we are evaluating the targeting properties of fusions made between a yeast sodium/proton exchanger (Sod2) and a proton pump (AHA2). This fusion may potentially lead to a new strategy for engineering salt resistant plants. Together these aims are designed to provide fundamental insights into the biogenesis and function of plant cell membrane systems

Final Report for DE-FG02-04ER15626: P-type ATPases in Plants – Role of Lipid Flippases in Membrane Biogenesis

Energy Technology Data Exchange (ETDEWEB)

Harper, Jeffrey F. [Univ. of Nevada, Reno, NV (United States)

2015-02-24

The long-range goal of the research is to understand the structure and biological functions of different P-type ATPases (ion pumps) in plant cells, and to use that knowledge to enhance the production of bioenergy from plants, or plant-research inspired technologies. Ptype ATPases include ion pumps that specifically transport H⁺, Ca²+, Zn²⁺, Cu²⁺, K⁺, or Na⁺, as well as at least one unusual subfamily that appears to function as lipid flippases, flipping specific lipids from one side of a membrane bilayer to the other. As a group, P-type ATPases are thought to consume more than 1/3 of the cellular ATP in typical eukaryotic cells. Recent research in the Harper lab focused on understanding the biochemical and biological functions of P-type ATPases that flip lipids. These flippases belong to the P4 subfamily of P-type ATPases. The activity of lipid flippases is thought to induce membrane curvature and/or create an asymmetry in which certain lipid head groups are preferential exposed to one surface or the other. In Arabidopsis thaliana there are 12 members of this family referred to as Aminophospholipid ATPase (ALA) 1 to ALA12. Using genetic knockouts, the Harper lab has established that this unusual subfamily of P-type ATPases are critical for plants to cope with even modest changes in temperature (e.g., down to 15°C, or up to 30°C). In addition, members of one subclade are critical for cell expansion, and loss of function mutants result in severe dwarfism. Other members of this same sub-clade are critical for pollen tube growth, and loss of function mutants are sterile under conditions of hot days and cold nights. While the cellular processes that depend on lipid flippases are still unclear, the genetic analysis of loss of function mutants clearly show they are of fundamental importance to plant growth and response to the environment.

Stabilization of the H,K-ATPase M5M6 membrane hairpin by K+ ions. Mechanistic significance for p2-type atpases.

Science.gov (United States)

Gatto, C; Lutsenko, S; Shin, J M; Sachs, G; Kaplan, J H

1999-05-14

The integral membrane protein, the gastric H,K-ATPase, is an alpha-beta heterodimer, with 10 putative transmembrane segments in the alpha-subunit and one such segment in the beta-subunit. All transmembrane segments remain within the membrane domain following trypsinization of the intact gastric H,K-ATPase in the presence of K+ ions, identified as M1M2, M3M4, M5M6, and M7, M8, M9, and M10. Removal of K+ ions from this digested preparation results in the selective loss of the M5M6 hairpin from the membrane. The release of the M5M6 fragment is directed to the extracellular phase as evidenced by the accumulation of the released M5M6 hairpin inside the sealed inside out vesicles. The stabilization of the M5M6 hairpin in the membrane phase by the transported cation as well as loss to the aqueous phase in the absence of the transported cation has been previously observed for another P2-type ATPase, the Na, K-ATPase (Lutsenko, S., Anderko, R., and Kaplan, J. H. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7936-7940). Thus, the effects of the counter-transported cation on retention of the M5M6 segment in the membrane as compared with the other membrane pairs may be a general feature of P2-ATPase ion pumps, reflecting a flexibility of this region that relates to the mechanism of transport.

Crystallization of P-type ATPases by the High Lipid-Detergent (HiLiDe) Method

DEFF Research Database (Denmark)

Sitsel, Oleg; Wang, Kaituo; Liu, Xiangyu

2016-01-01

Determining structures of membrane proteins remains a significant challenge. A technique utilizing high lipid-detergent concentrations ("HiLiDe") circumvents the major bottlenecks of current membrane protein crystallization methods. During HiLiDe, the protein-lipid-detergent ratio is varied in a ...... crystallization techniques. The method has been applied with particular success to P-type ATPases....

The Role of the N-Domain in the ATPase Activity of the Mammalian AAA ATPase p97/VCP*

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Niwa, Hajime; Ewens, Caroline A.; Tsang, Chun; Yeung, Heidi O.; Zhang, Xiaodong; Freemont, Paul S.

2012-01-01

p97/valosin-containing protein (VCP) is a type II ATPase associated with various cellular activities that forms a homohexamer with each protomer containing an N-terminal domain (N-domain); two ATPase domains, D1 and D2; and a disordered C-terminal region. Little is known about the role of the N-domain or the C-terminal region in the p97 ATPase cycle. In the p97-associated human disease inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia, the majority of missense mutations are located at the N-domain D1 interface. Structure-based predictions suggest that such mutations affect the interaction of the N-domain with D1. Here we have tested ten major inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia-linked mutants for ATPase activity and found that all have increased activity over the wild type, with one mutant, p97A232E, having three times higher activity. Further mutagenesis of p97A232E shows that the increase in ATPase activity is mediated through D2 and requires both the N-domain and a flexible ND1 linker. A disulfide mutation that locks the N-domain to D1 in a coplanar position reversibly abrogates ATPase activity. A cryo-EM reconstruction of p97A232E suggests that the N-domains are flexible. Removal of the C-terminal region also reduces ATPase activity. Taken together, our data suggest that the conformation of the N-domain in relation to the D1-D2 hexamer is directly linked to ATP hydrolysis and that the C-terminal region is required for hexamer stability. This leads us to propose a model where the N-domain adopts either of two conformations: a flexible conformation compatible with ATP hydrolysis or a coplanar conformation that is inactive. PMID:22270372

The role of the N-domain in the ATPase activity of the mammalian AAA ATPase p97/VCP.

Science.gov (United States)

Niwa, Hajime; Ewens, Caroline A; Tsang, Chun; Yeung, Heidi O; Zhang, Xiaodong; Freemont, Paul S

2012-03-09

p97/valosin-containing protein (VCP) is a type II ATPase associated with various cellular activities that forms a homohexamer with each protomer containing an N-terminal domain (N-domain); two ATPase domains, D1 and D2; and a disordered C-terminal region. Little is known about the role of the N-domain or the C-terminal region in the p97 ATPase cycle. In the p97-associated human disease inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia, the majority of missense mutations are located at the N-domain D1 interface. Structure-based predictions suggest that such mutations affect the interaction of the N-domain with D1. Here we have tested ten major inclusion body myopathy associated with Paget disease of bone and frontotemporal dementia-linked mutants for ATPase activity and found that all have increased activity over the wild type, with one mutant, p97(A232E), having three times higher activity. Further mutagenesis of p97(A232E) shows that the increase in ATPase activity is mediated through D2 and requires both the N-domain and a flexible ND1 linker. A disulfide mutation that locks the N-domain to D1 in a coplanar position reversibly abrogates ATPase activity. A cryo-EM reconstruction of p97(A232E) suggests that the N-domains are flexible. Removal of the C-terminal region also reduces ATPase activity. Taken together, our data suggest that the conformation of the N-domain in relation to the D1-D2 hexamer is directly linked to ATP hydrolysis and that the C-terminal region is required for hexamer stability. This leads us to propose a model where the N-domain adopts either of two conformations: a flexible conformation compatible with ATP hydrolysis or a coplanar conformation that is inactive.

Cardiac glycoside activities link Na(+)/K(+) ATPase ion-transport to breast cancer cell migration via correlative SAR.

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Magpusao, Anniefer N; Omolloh, George; Johnson, Joshua; Gascón, José; Peczuh, Mark W; Fenteany, Gabriel

2015-02-20

The cardiac glycosides ouabain and digitoxin, established Na(+)/K(+) ATPase inhibitors, were found to inhibit MDA-MB-231 breast cancer cell migration through an unbiased chemical genetics screen for cell motility. The Na(+)/K(+) ATPase acts both as an ion-transporter and as a receptor for cardiac glycosides. To delineate which function is related to breast cancer cell migration, structure-activity relationship (SAR) profiles of cardiac glycosides were established at the cellular (cell migration inhibition), molecular (Na(+)/K(+) ATPase inhibition), and atomic (computational docking) levels. The SAR of cardiac glycosides and their analogs revealed a similar profile, a decrease in potency when the parent cardiac glycoside structure was modified, for each activity investigated. Since assays were done at the cellular, molecular, and atomic levels, correlation of SAR profiles across these multiple assays established links between cellular activity and specific protein-small molecule interactions. The observed antimigratory effects in breast cancer cells are directly related to the inhibition of Na(+)/K(+) transport. Specifically, the orientation of cardiac glycosides at the putative cation permeation path formed by transmembrane helices αM1-M6 correlates with the Na(+) pump activity and cell migration. Other Na(+)/K(+) ATPase inhibitors that are structurally distinct from cardiac glycosides also exhibit antimigratory activity, corroborating the conclusion that the antiport function of Na(+)/K(+) ATPase and not the receptor function is important for supporting the motility of MDA-MB-231 breast cancer cells. Correlative SAR can establish new relationships between specific biochemical functions and higher-level cellular processes, particularly for proteins with multiple functions and small molecules with unknown or various modes of action.

Iron overload impact on P-ATPases.

Science.gov (United States)

Sousa, Leilismara; Pessoa, Marco Tulio C; Costa, Tamara G F; Cortes, Vanessa F; Santos, Herica L; Barbosa, Leandro Augusto

2018-03-01

Iron is a chemical element that is active in the fundamental physiological processes for human life, but its burden can be toxic to the body, mainly because of the stimulation of membrane lipid peroxidation. For this reason, the action of iron on many ATPases has been studied, especially on P-ATPases, such as the Na + ,K + -ATPase and the Ca 2+ -ATPase. On the Fe 2+ -ATPase activity, the free iron acts as an activator, decreasing the intracellular Fe 2+ and playing a protection role for the cell. On the Ca 2+ -ATPase activity, the iron overload decreases the enzyme activity, raising the cytoplasmic Ca 2+ and decreasing the sarco/endoplasmic reticulum and the Golgi apparatus Ca 2+ concentrations, which could promote an enzyme oxidation, nitration, and fragmentation. However, the iron overload effect on the Na + ,K + -ATPase may change according to the tissue expressions. On the renal cells, as well as on the brain and the heart, iron promotes an enzyme inactivation, whereas its effect on the erythrocytes seems to be the opposite, directly stimulating the ATPase activity, or stimulating it by signaling pathways involving ROS and PKC. Modulations in the ATPase activity may impair the ionic transportation, which is essential for cell viability maintenance, inducing irreversible damage to the cell homeostasis. Here, we will discuss about the iron overload effect on the P-ATPases, such as the Na + ,K + -ATPase, the Ca 2+ -ATPase, and the Fe 2+ -ATPase.

Novel Sulfur Metabolites of Garlic Attenuate Cardiac Hypertrophy and Remodeling through Induction of Na+/K+-ATPase Expression.

Science.gov (United States)

Khatua, Tarak N; Borkar, Roshan M; Mohammed, Soheb A; Dinda, Amit K; Srinivas, R; Banerjee, Sanjay K

2017-01-01

Epidemiologic studies show an inverse correlation between garlic consumption and progression of cardiovascular disease. However, the molecular basis for the beneficial effect of garlic on the heart is not known. Therefore, the objective of the present study was to (1) investigate the effect of raw garlic on isoproterenol (Iso) induced cardiac hypertrophy (2) find the active metabolites of garlic responsible for the beneficial effect. Cardiac hypertrophy was induced in rats by subcutaneous single injection of Iso 5 mg kg -1 day -1 for 15 days and the effect of garlic (250 mg/kg/day orally) was evaluated. Garlic metabolites in in vivo were identified by LC/MS study. The effect of garlic and its metabolites were evaluated against hypertrophy in H9C2 cells. Garlic normalized cardiac oxidative stress after Iso administration. Cardiac pathology and mitochondrial enzyme activities were improved in hypertrophy heart after garlic administration. Decreased Na + /K + -ATPase protein level that observed in hypertrophy heart was increased after garlic administration. We identified three garlic metabolites in rat serum. To confirm the role of garlic metabolites on cardiac hypertrophy, Na + /K + -ATPase expression and intracellular calcium levels were measured after treating H9C2 cells with raw garlic and two of its active metabolites, allyl methyl sulfide and allyl methyl sulfoxide. Raw garlic and both metabolites increased Na + /K + -ATPase protein level and decreased intracellular calcium levels and cell size in Iso treated H9C2 cells. This antihypertrophic effect of garlic and its sulfur metabolites were lost in H9C2 cells in presence of Na + /K + -ATPase inhibitor. In conclusion, garlic and its active metabolites increased Na + /K + -ATPase in rat heart, and attenuated cardiac hypertrophy and associated remodeling. Our data suggest that identified new garlic metabolites may be useful for therapeutic intervention against cardiac hypertrophy.

Oleic and linoleic acids are active principles in Nigella sativa and stabilize an E(2)P conformation of the Na,K-ATPase. Fatty acids differentially regulate cardiac glycoside interaction with the pump.

Science.gov (United States)

Mahmmoud, Yasser A; Christensen, S Brøgger

2011-10-01

Nigella sativa seed oil was found to contain a modulator of Na,K-ATPase. Separation analyses combined with (1)H NMR and GCMS identified the inhibitory fraction as a mixture of oleic and linoleic acids. These two fatty acids are specifically concentrated in several medicinal plant oils, and have particularly been implicated in decreasing high blood pressure. The ouabain binding site on Na,K-ATPase has also been implicated in blood pressure regulation. Thus, we aimed to determine how these two molecules modify pig kidney Na,K-ATPase. Oleic and linoleic acids did not modify reactions involving the E(1) (Na(+)) conformations of the Na,K-ATPase. In contrast, K(+) dependent reactions were strongly modified after treatment. Oleic and linoleic acids were found to stabilize a pump conformation that binds ouabain with high affinity, i.e., an ion free E(2)P form. Time-resolved binding assays using anthroylouabain, a fluorescent ouabain analog, revealed that the increased ouabain affinity is unique to oleic and linoleic acids, as compared with γ-linolenic acid, which decreased pump-mediated ATP hydrolysis but did not equally increase ouabain interaction with the pump. Thus, the dynamic changes in plasma levels of oleic and linoleic acids are important in the modulation of the sensitivity of the sodium pump to cardiac glycosides. Given the possible involvement of the cardiac glycoside binding site on Na,K-ATPase in the regulation of hypertension, we suggest oleic acid to be a specific chaperon that modulates interaction of cardiac glycosides with the sodium pump. Crown Copyright © 2011. Published by Elsevier B.V. All rights reserved.

TRANSPARENT TESTA 13 is a tonoplast P3A -ATPase required for vacuolar deposition of proanthocyanidins in Arabidopsis thaliana seeds

NARCIS (Netherlands)

Appelhagen, I.; Nordholt, N.; Seidel, T.; Spelt, K.; Koes, R.; Quattrochio, F.; Sagasser, M.; Weisshaar, B.

2015-01-01

Intracellular pH homeostasis is essential for all living cells. In plants, pH is usually maintained by three structurally distinct and differentially localized types of proton pump: P-type H(+) -ATPases in the plasma membrane, and multimeric vacuolar-type H(+) -ATPases (V-ATPases) and vacuolar H(+)

Mechanism and significance of P4 ATPase-catalyzed lipid transport: lessons from a Na+/K+-pump

NARCIS (Netherlands)

Puts, C.F.; Holthuis, J.C.M.

2009-01-01

Members of the P4 subfamily of P-type ATPases are believed to catalyze phospholipid transport across membrane bilayers, a process influencing a host of cellular functions. Atomic structures and functional analysis of P-type ATPases that pump small cations and metal ions revealed a transport

Structural models of the human copper P-type ATPases ATP7A and ATP7B

DEFF Research Database (Denmark)

Gourdon, P.; Sitsel, Oleg; Karlsen, J.L.

2012-01-01

The human copper exporters ATP7A and ATP7B contain domains common to all P-type ATPases as well as class-specific features such as six sequential heavy-metal binding domains (HMBD1-HMBD6) and a type-specific constellation of transmembrane helices. Despite the medical significance of ATP7A and ATP7B......, allowing protein-specific properties to be addressed. Furthermore, the mapping of known disease-causing missense mutations indicates that among the heavy-metal binding domains, HMBD5 and HMBD6 are the most crucial for function, thus mimicking the single or dual HMBDs found in most copper-specific P-type...

Dependence of myosin-ATPase on structure bound creatine kinase in cardiac myfibrils from rainbow trout and freshwater turtle

DEFF Research Database (Denmark)

Haagensen, L.; Jensen, D.H.; Gesser, Hans

2008-01-01

The influence of myofibrillar creatine kinase on the myosin-ATPase activity was examined in cardiac ventricular myofibrils isolated from rainbow trout (Oncorhynchus mykiss) and freshwater turtle (Trachemys scripta). The ATPase rate was assessed by recording the rephosphorylation of ADP by the pyr......The influence of myofibrillar creatine kinase on the myosin-ATPase activity was examined in cardiac ventricular myofibrils isolated from rainbow trout (Oncorhynchus mykiss) and freshwater turtle (Trachemys scripta). The ATPase rate was assessed by recording the rephosphorylation of ADP...... by the pyruvate kinase reaction alone or together with the amount of creatine formed, when myofibrillar bound creatine kinase was activated with phosphocreatine. The steady-state concentration of ADP in the solution was varied through the activity of pyruvate kinase added to the solution. For rainbow trout...... myofibrils at a high pyruvate kinase activity, creatine kinase competed for ADP but did not influence the total ATPase activity. When the ADP concentration was elevated within the physiological range by lowering the pyruvate kinase activity, creatine kinase competed efficiently and increased the ATPase...

P4 ATPases: Flippases in Health and Disease

Directory of Open Access Journals (Sweden)

Coen C. Paulusma

2013-04-01

Full Text Available P4 ATPases catalyze the translocation of phospholipids from the exoplasmic to the cytosolic leaflet of biological membranes, a process termed “lipid flipping”. Accumulating evidence obtained in lower eukaryotes points to an important role for P4 ATPases in vesicular protein trafficking. The human genome encodes fourteen P4 ATPases (fifteen in mouse of which the cellular and physiological functions are slowly emerging. Thus far, deficiencies of at least two P4 ATPases, ATP8B1 and ATP8A2, are the cause of severe human disease. However, various mouse models and in vitro studies are contributing to our understanding of the cellular and physiological functions of P4-ATPases. This review summarizes current knowledge on the basic function of these phospholipid translocating proteins, their proposed action in intracellular vesicle transport and their physiological role.

New ATPase regulators-p97 goes to the PUB

DEFF Research Database (Denmark)

Madsen, Louise; Seeger, Michael; Semple, Colin A

2009-01-01

The conserved eukaryotic AAA-type ATPase complex, known as p97 or VCP in mammals and Cdc48 in yeast, is involved in a number of cellular pathways, including fusion of homotypic membranes, protein degradation, and activation of membrane-bound transcription factors. Most likely, p97 is directed...

Disease mutations reveal residues critical to the interaction of P4-ATPases with lipid substrates

DEFF Research Database (Denmark)

Gantzel, Rasmus H; Mogensen, Louise S; Mikkelsen, Stine A

2017-01-01

Phospholipid flippases (P4-ATPases) translocate specific phospholipids from the exoplasmic to the cytoplasmic leaflet of membranes. While there is good evidence that the overall molecular structure of flippases is similar to that of P-type ATPase ion-pumps, the transport pathway for the "giant...

Kinase-Mediated Regulation of P4-ATPases

DEFF Research Database (Denmark)

Frøsig, Merethe Mørch

designed a fast and efficient screening strategy to identify novel regulator proteins of P4-ATPases. The system is based on heterologous expression in a specially designed yeast strain, and regulatory proteins can be identified via change in activity of the P4-ATPase of interest. Hereby the first steps...

Functional Analysis of P4-ATPases

DEFF Research Database (Denmark)

Theorin, Lisa

and mammalian P4-ATPases have been studied extensively and the physiological function is mostly known, while the exact biochemistry and specific activity is mostly unknown. Even though the plant Arabidopsis thaliana has 12 P4-ATPases, not much is known about their function. In this study, the biochemical...... for purification of the complex by one-step purification. The ATPase activity of the ALA2/ALIS5 complex was stimulated in a highly specific manner by phosphatidylserine. Changes in the phosphatidylserine headgroup or alteration of the stereochemistry affected enzymatic activity. The results demonstrate that ALA2...... is specific for phosphatidylserine and that binding of the lipid to the substrate binding site requires a unique spatial configuration of the lipid head group. Detailed information on the substrate requirements lead the way towards the full function and transport pathway of lipid flippases in plants. Recent...

Review: P4-ATPases as Phospholipid Flippases-Structure, Function, and Enigmas

DEFF Research Database (Denmark)

Andersen, Jens P; Vestergaard, Anna L; Mikkelsen, Stine A

2016-01-01

group is propelled along against its concentration gradient with the hydrocarbon chains projecting out into the lipid phase by movement of an isoleucine located at the position corresponding to an ion binding glutamate in the Ca2+- and Na+/K+-ATPases. Hence, the P4-ATPase mechanism is quite similar...... on properties of mammalian and yeast P4-ATPases for which most mechanistic insight is available. However, the structure, function and enigmas associated with mammalian and yeast P4-ATPases most likely extend to P4-ATPases of plants and other organisms....

Characterization of a heavy metal translocating P-type ATPase gene from an environmental heavy metal resistance Enterobacter sp. isolate.

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Chien, Chih-Ching; Huang, Chia-Hsuan; Lin, Yi-Wei

2013-03-01

Heavy metals are common contaminants found in polluted areas. We have identified a heavy metal translocating P-type ATPase gene (hmtp) via fosmid library and in vitro transposon mutagenesis from an Enterobacter sp. isolate. This gene is believed to participate in the bacterium's heavy metal resistance traits. The complete gene was identified, cloned, and expressed in a suitable Escherichia coli host cell. E. coli W3110, RW3110 (zntA::Km), GG48 (ΔzitB::Cm zntA::Km), and GG51 (ΔzitB::Cm) were used to study the possible effects of this gene for heavy metal (cadmium and zinc in particular) resistance. Among the E. coli strains tested, RW3110 and GG48 showed more sensitivity to cadmium and zinc compared to the wild-type E. coli W3110 and strain GG51. Therefore, strains RW3110 and GG48 were chosen for the reference hosts for further evaluation of the gene's effect. The results showed that expression of this heavy metal translocating P-type ATPase gene could increase the ability for zinc and cadmium resistance in the tested microorganisms.

P4-ATPases on the spotlight: lessons from a green world

DEFF Research Database (Denmark)

Lopez Marques, Rosa Laura

flippases, play an essential role in this transport process. We have recently characterized several members of the P4 subfamily of P-type ATPases as prime candidate lipid flippases in the secretory pathway of several eukaryotic cells. Our studies in yeast, plants and mammalian cells uncovered...

Membrane Anchoring and Ion-Entry Dynamics in P-type ATPase Copper Transport

DEFF Research Database (Denmark)

Grønberg, Christina; Sitsel, Oleg; Lindahl, Erik

2016-01-01

Cu(+)-specific P-type ATPase membrane protein transporters regulate cellular copper levels. The lack of crystal structures in Cu(+)-binding states has limited our understanding of how ion entry and binding are achieved. Here, we characterize the molecular basis of Cu(+) entry using molecular-dynamics...... simulations, structural modeling, and in vitro and in vivo functional assays. Protein structural rearrangements resulting in the exposure of positive charges to bulk solvent rather than to lipid phosphates indicate a direct molecular role of the putative docking platform in Cu(+) delivery. Mutational analyses...... and simulations in the presence and absence of Cu(+) predict that the ion-entry path involves two ion-binding sites: one transient Met148-Cys382 site and one intramembranous site formed by trigonal coordination to Cys384, Asn689, and Met717. The results reconcile earlier biochemical and x-ray absorption data...

Vacuolar ATPase regulates surfactant secretion in rat alveolar type II cells by modulating lamellar body calcium.

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Narendranath Reddy Chintagari

2010-02-01

Full Text Available Lung surfactant reduces surface tension and maintains the stability of alveoli. How surfactant is released from alveolar epithelial type II cells is not fully understood. Vacuolar ATPase (V-ATPase is the enzyme responsible for pumping H(+ into lamellar bodies and is required for the processing of surfactant proteins and the packaging of surfactant lipids. However, its role in lung surfactant secretion is unknown. Proteomic analysis revealed that vacuolar ATPase (V-ATPase dominated the alveolar type II cell lipid raft proteome. Western blotting confirmed the association of V-ATPase a1 and B1/2 subunits with lipid rafts and their enrichment in lamellar bodies. The dissipation of lamellar body pH gradient by Bafilomycin A1 (Baf A1, an inhibitor of V-ATPase, increased surfactant secretion. Baf A1-stimulated secretion was blocked by the intracellular Ca(2+ chelator, BAPTA-AM, the protein kinase C (PKC inhibitor, staurosporine, and the Ca(2+/calmodulin-dependent protein kinase II (CaMKII, KN-62. Baf A1 induced Ca(2+ release from isolated lamellar bodies. Thapsigargin reduced the Baf A1-induced secretion, indicating cross-talk between lamellar body and endoplasmic reticulum Ca(2+ pools. Stimulation of type II cells with surfactant secretagogues dissipated the pH gradient across lamellar bodies and disassembled the V-ATPase complex, indicating the physiological relevance of the V-ATPase-mediated surfactant secretion. Finally, silencing of V-ATPase a1 and B2 subunits decreased stimulated surfactant secretion, indicating that these subunits were crucial for surfactant secretion. We conclude that V-ATPase regulates surfactant secretion via an increased Ca(2+ mobilization from lamellar bodies and endoplasmic reticulum, and the activation of PKC and CaMKII. Our finding revealed a previously unrealized role of V-ATPase in surfactant secretion.

Relationship between serum adiponectin level and ATPase activity of erythrocyte membrance in patients with 2-type diabetes

International Nuclear Information System (INIS)

Song Jiejin

2008-01-01

Objective: To explore the possible mechanism of development nephrosis as related to changes of serum adiponectin levels and alteration of activities of Na + ·K + -ATPase and Ca +2 ·Mg +2 -ATPase of erythrocyte membrance in patients with 2-type diabetes. Methods: Serum adiponectin levels (with RIA) and erythrocyte membrance (prepared with Reilnila method) Na + ·K + - ATPase and Ca +2 ·Mg +2 -ATPase activity were determined in 45 DM2 patients without nephropathy, 31 DM2 patients with nephropathy and 30 controls. Results: Serum adiponectin levels in the diabetic patients were significantly lower than those in controls (P + ·K + -ATPase and Ca +2 ·Mg +2 -ATPase activities were also significantly lower than those in controls (P + ·K + -ATPase and Ca +2 ·Mg +2 -ATPase activities of erythrocyte membrance. (authors)

Cloning, purification and crystallization of a Walker-type Pyrococcus abyssi ATPase family member

International Nuclear Information System (INIS)

Uhring, Muriel; Bey, Gilbert; Lecompte, Odile; Cavarelli, Jean; Moras, Dino; Poch, Olivier

2005-01-01

The Walker-type ATPase PABY2304 of P. abyssi has been cloned, overexpressed, purified and crystallized. X-ray diffraction data from selenomethionine-derivative crystals have been collected to 2.6 Å. The structure has been solved by MAD techniques. Several ATPase proteins play essential roles in the initiation of chromosomal DNA replication in archaea. Walker-type ATPases are defined by their conserved Walker A and B motifs, which are associated with nucleotide binding and ATP hydrolysis. A family of 28 ATPase proteins with non-canonical Walker A sequences has been identified by a bioinformatics study of comparative genomics in Pyrococcus genomes. A high-throughput structural study on P. abyssi has been started in order to establish the structure of these proteins. 16 genes have been cloned and characterized. Six out of the seven soluble constructs were purified in Escherichia coli and one of them, PABY2304, has been crystallized. X-ray diffraction data were collected from selenomethionine-derivative crystals using synchrotron radiation. The crystals belong to the orthorhombic space group C2, with unit-cell parameters a = 79.41, b = 48.63, c = 108.77 Å, and diffract to beyond 2.6 Å resolution

Cloning, purification and crystallization of a Walker-type Pyrococcus abyssi ATPase family member

Energy Technology Data Exchange (ETDEWEB)

Uhring, Muriel; Bey, Gilbert; Lecompte, Odile; Cavarelli, Jean; Moras, Dino; Poch, Olivier, E-mail: poch@igbmc.u-strasbg.fr [Département de Biologie et Génomiques Structurales, UMR 7104, Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP Strasbourg, 1 Rue Laurent Fries, 64404 Illkirch (France)

2005-10-01

The Walker-type ATPase PABY2304 of P. abyssi has been cloned, overexpressed, purified and crystallized. X-ray diffraction data from selenomethionine-derivative crystals have been collected to 2.6 Å. The structure has been solved by MAD techniques. Several ATPase proteins play essential roles in the initiation of chromosomal DNA replication in archaea. Walker-type ATPases are defined by their conserved Walker A and B motifs, which are associated with nucleotide binding and ATP hydrolysis. A family of 28 ATPase proteins with non-canonical Walker A sequences has been identified by a bioinformatics study of comparative genomics in Pyrococcus genomes. A high-throughput structural study on P. abyssi has been started in order to establish the structure of these proteins. 16 genes have been cloned and characterized. Six out of the seven soluble constructs were purified in Escherichia coli and one of them, PABY2304, has been crystallized. X-ray diffraction data were collected from selenomethionine-derivative crystals using synchrotron radiation. The crystals belong to the orthorhombic space group C2, with unit-cell parameters a = 79.41, b = 48.63, c = 108.77 Å, and diffract to beyond 2.6 Å resolution.

Intracellular pH (pHin) and cytosolic calcium ([Ca2+]cyt) regulation via ATPases: studies in cell populations, single cells, and subcellular compartments

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Rojas, Jose D.; Sanka, Shankar C.; Gyorke, Sandor; Wesson, Donald E.; Minta, Akwasi; Martinez-Zaguilan, Raul

1999-07-01

Changes in pHin and (Ca2+)cyt are important in the signal transduction mechanisms leading to many physiological responses including cell growth, motility, secretion/exocytosis, etc. The concentrations of these ions are regulated via primary and secondary ion transporting mechanisms. In diabetes, specific pH and Ca2+ regulatory mechanism might be altered. To study these ions, we employ fluorescence spectroscopy, and cell imagin spectroscopy/confocal microscopy. pH and Ca2+ indicators are loaded in the cytosol with acetoxymethyl ester forms of dyes, and in endosomal/lysosomal (E/L) compartments by overnight incubation of cells with dextran- conjugated ion fluorescent probes. We focus on specific pH and Ca2+ regulatory systems: plasmalemmal vacuolar- type H+-ATPases (pm V-ATPases) and sarcoplasmic/endoplasmic reticulum Ca2+-ATPases (SERCA). As experimental models, we employ vascular smooth muscle (VSM) and microvascular endothelial cells. We have chosen these cells because they are important in blood flow regulation and in angiogenesis. These processes are altered in diabetes. In many cell types, ion transport processes are dependent on metabolism of glucose for maximal activity. Our main findings are: (a) glycolysis coupling the activity of SERCA is required for cytosolic Ca2+ homeostasis in both VSM and microvascular endothelial cells; (b) E/L compartments are important for pH and Ca2+ regulation via H+-ATPases and SERCA, respectively; and (c) pm-V- ATPases are important for pHin regulation in microvascular endothelial cells.

Intracellular pH homeostasis and serotonin-induced pH changes in Calliphora salivary glands: the contribution of V-ATPase and carbonic anhydrase.

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Schewe, Bettina; Schmälzlin, Elmar; Walz, Bernd

2008-03-01

Blowfly salivary gland cells have a vacuolar-type H(+)-ATPase (V-ATPase) in their apical membrane that energizes secretion of a KCl-rich saliva upon stimulation with serotonin (5-hydroxytryptamine, 5-HT). We have used BCECF to study microfluometrically whether V-ATPase and carbonic anhydrase (CA) are involved in intracellular pH (pH(i)) regulation, and we have localized CA activity by histochemistry. We show: (1) mean pH(i) in salivary gland cells is 7.5+/-0.3 pH units (N=96), higher than that expected from passive H(+) distribution; (2) low 5-HT concentrations (0.3-3 nmol l(-1)) induce a dose-dependent acidification of up to 0.2 pH units, with 5-HT concentrations >10 nmol l(-1), causing monophasic or multiphasic pH changes; (3) the acidifying effect of 5-HT is mimicked by bath application of cAMP, forskolin or IBMX; (4) salivary gland cells exhibit CA activity; (5) CA inhibition with acetazolamide and V-ATPase inhibition with concanamycin A lead to a slow acidification of steady-state pH(i); (6) 5-HT stimuli in the presence of acetazolamide induce an alkalinization that can be decreased by simultaneous application of the V-ATPase inhibitor concanamycin A; (7) concanamycin A removes alkali-going components from multiphasic 5-HT-induced pH changes; (8) NHE activity and a Cl(-)-dependent process are involved in generating 5-HT-induced pH changes; (9) the salivary glands probably contain a Na(+)-driven amino acid transporter. We conclude that V-ATPase and CA contribute to steady-state pH(i) regulation and 5-HT-induced outward H(+) pumping does not cause an alkalinization of pH(i) because of cytosolic H(+) accumulation attributable to stimulated cellular respiration and AE activity, masking the alkalizing effect of V-ATPase-mediated acid extrusion.

Intracellular pH regulation in unstimulated Calliphora salivary glands is Na+ dependent and requires V-ATPase activity.

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Schewe, Bettina; Blenau, Wolfgang; Walz, Bernd

2012-04-15

Salivary gland cells of the blowfly Calliphora vicina have a vacuolar-type H(+)-ATPase (V-ATPase) that lies in their apical membrane and energizes the secretion of a KCl-rich primary saliva upon stimulation with serotonin (5-hydroxytryptamine). Whether and to what extent V-ATPase contributes to intracellular pH (pH(i)) regulation in unstimulated gland cells is unknown. We used the fluorescent dye BCECF to study intracellular pH(i) regulation microfluorometrically and show that: (1) under resting conditions, the application of Na(+)-free physiological saline induces an intracellular alkalinization attributable to the inhibition of the activity of a Na(+)-dependent glutamate transporter; (2) the maintenance of resting pH(i) is Na(+), Cl(-), concanamycin A and DIDS sensitive; (3) recovery from an intracellular acid load is Na(+) sensitive and requires V-ATPase activity; (4) the Na(+)/H(+) antiporter is not involved in pH(i) recovery after a NH(4)Cl prepulse; and (5) at least one Na(+)-dependent transporter and the V-ATPase maintain recovery from an intracellular acid load. Thus, under resting conditions, the V-ATPase and at least one Na(+)-dependent transporter maintain normal pH(i) values of pH 7.5. We have also detected the presence of a Na(+)-dependent glutamate transporter, which seems to act as an acid loader. Despite this not being a common pH(i)-regulating transporter, its activity affects steady-state pH(i) in C. vicina salivary gland cells.

Regulatory Mechanisms in the P4-ATPase Complex

DEFF Research Database (Denmark)

Costa, Sara

. The functionality on the P4-ATPase complex is essential for several cellular processes, such as vesicle-mediated transport. However, the specific role of flippase activity in vesicle biogenesis and the regulatory mechanism behind this process is still poorly understood. In these studies, we identified...... affordable alternative using a microscope-based cytometer. This system can simultaneously provide information on flippase activity and expression levels. Taken together, the findings described in this thesis provide new tools for P4-ATPase characterization and valuable insights into the regulation...

Direct interaction of the Golgi V-ATPase a-subunit isoform with PI(4)P drives localization of Golgi V-ATPases in yeast.

Science.gov (United States)

Banerjee, Subhrajit; Kane, Patricia M

2017-09-15

Luminal pH and phosphoinositide content are fundamental features of organelle identity. Vacuolar H + -ATPases (V-ATPases) drive organelle acidification in all eukaryotes, and membrane-bound a-subunit isoforms of the V-ATPase are implicated in organelle-specific targeting and regulation. Earlier work demonstrated that the endolysosomal lipid PI(3,5)P 2 activates V-ATPases containing the vacuolar a-subunit isoform in Saccharomyces cerevisiae Here we demonstrate that PI(4)P, the predominant Golgi phosphatidylinositol (PI) species, directly interacts with the cytosolic amino terminal (NT) domain of the yeast Golgi V-ATPase a-isoform Stv1. Lysine-84 of Stv1NT is essential for interaction with PI(4)P in vitro and in vivo, and interaction with PI(4)P is required for efficient localization of Stv1-containing V-ATPases. The cytosolic NT domain of the human V-ATPase a2 isoform specifically interacts with PI(4)P in vitro, consistent with its Golgi localization and function. We propose that NT domains of V o a-subunit isoforms interact specifically with PI lipids in their organelles of residence. These interactions can transmit organelle-specific targeting or regulation information to V-ATPases. © 2017 Banerjee and Kane. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

A cloned prokaryotic Cd2+ P-type ATPase increases yeast sensitivity to Cd2+

International Nuclear Information System (INIS)

Wu, C.-C.; Bal, Nathalie; Perard, Julien; Lowe, Jennifer; Boscheron, Cecile; Mintz, Elisabeth; Catty, Patrice

2004-01-01

CadA, the P1-type ATPase involved in Listeria monocytogenes resistance to Cd 2+ , was expressed in Saccharomyces cerevisiae and did just the opposite to what was expected, as it strikingly decreased the Cd 2+ tolerance of these cells. Yeast cells expressing the non-functional mutant Asp 398 Ala could grow on selective medium containing up to 100 μM Cd 2+ , whereas those expressing the functional protein could not grow in the presence of 1 μM Cd 2+ . The CadA-GFP fusion protein was localized in the endoplasmic reticulum membrane, suggesting that yeast hyper-sensitivity was due to Cd 2+ accumulation in the reticulum lumen. CadA is also known to transport Zn 2+ , but Zn 2+ did not protect the cells against Cd 2+ poisoning. In the presence of 10 μM Cd 2+ , transformed yeasts survived by rapid loss of their expression vector

Nitric oxide and Na,K-ATPase activity in rat skeletal muscle.

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Juel, C

2016-04-01

It has been suggested that nitric oxide (NO) stimulates the Na,K-ATPase in cardiac myocytes. Therefore, the aims of this study were to investigate whether NO increases Na,K-ATPase activity in skeletal muscle and, if that is the case, to identify the underlying mechanism. The study used isolated rat muscle, muscle homogenates and purified membranes as model systems. Na,K-ATPase activity was quantified from phosphate release due to ATP hydrolysis. Exposure to the NO donor spermine NONOate (10 μm) increased the maximal Na,K-ATPase activity by 27% in isolated glycolytic muscles, but had no effect in oxidative muscles. Spermine NONOate increased the maximal Na,K-ATPase activity by 58% (P Na,K-ATPase α-isoform. Incubation with cGMP (1 mm) increased the maximal Na,K-ATPase activity in homogenates from glycolytic muscle by 16% (P Na,K-ATPase in glycolytic skeletal muscle. Direct S-nitrosylation and interference with S-glutathionylation seem to be excluded. In addition, phosphorylation of phospholemman at serine 68 is not involved. Most likely, the NO/cGMP/protein kinase G signalling pathway is involved. © 2015 Scandinavian Physiological Society. Published by John Wiley & Sons Ltd.

The V-ATPase a2-subunit as a putative endosomal pH-sensor.

Science.gov (United States)

Marshansky, V

2007-11-01

V-ATPase (vesicular H(+)-ATPase)-driven intravesicular acidification is crucial for vesicular trafficking. Defects in vesicular acidification and trafficking have recently been recognized as essential determinants of various human diseases. An important role of endosomal acidification in receptor-ligand dissociation and in activation of lysosomal hydrolytic enzymes is well established. However, the molecular mechanisms by which luminal pH information is transmitted to the cytosolic small GTPases that control trafficking events such as budding, coat formation and fusion are unknown. Here, we discuss our recent discovery that endosomal V-ATPase is a pH-sensor regulating the degradative pathway. According to our model, V-ATPase is responsible for: (i) the generation of a pH gradient between vesicular membranes; (ii) sensing of intravesicular pH; and (iii) transmitting this information to the cytosolic side of the membrane. We also propose the hypothetical molecular mechanism involved in function of the V-ATPase a2-subunit as a putative pH-sensor. Based on extensive experimental evidence on the crucial role of histidine residues in the function of PSPs (pH-sensing proteins) in eukaryotic cells, we hypothesize that pH-sensitive histidine residues within the intra-endosomal loops and/or C-terminal luminal tail of the a2-subunit could also be involved in the pH-sensing function of V-ATPase. However, in order to identify putative pH-sensitive histidine residues and to test this hypothesis, it is absolutely essential that we increase our understanding of the folding and transmembrane topology of the a-subunit isoforms of V-ATPase. Thus the crucial role of intra-endosomal histidine residues in pH-dependent conformational changes of the V-ATPase a2-isoform, its interaction with cytosolic small GTPases and ultimately in its acidification-dependent regulation of the endosomal/lysosomal protein degradative pathway remain to be determined.

Molecular architecture of the N-type ATPase rotor ring from Burkholderia pseudomallei.

Science.gov (United States)

Schulz, Sarah; Wilkes, Martin; Mills, Deryck J; Kühlbrandt, Werner; Meier, Thomas

2017-04-01

The genome of the highly infectious bacterium Burkholderia pseudomallei harbors an atp operon that encodes an N-type rotary ATPase, in addition to an operon for a regular F-type rotary ATPase. The molecular architecture of N-type ATPases is unknown and their biochemical properties and cellular functions are largely unexplored. We studied the B. pseudomallei N 1 N o -type ATPase and investigated the structure and ion specificity of its membrane-embedded c-ring rotor by single-particle electron cryo-microscopy. Of several amphiphilic compounds tested for solubilizing the complex, the choice of the low-density, low-CMC detergent LDAO was optimal in terms of map quality and resolution. The cryoEM map of the c-ring at 6.1 Å resolution reveals a heptadecameric oligomer with a molecular mass of ~141 kDa. Biochemical measurements indicate that the c 17 ring is H + specific, demonstrating that the ATPase is proton-coupled. The c 17 ring stoichiometry results in a very high ion-to-ATP ratio of 5.7. We propose that this N-ATPase is a highly efficient proton pump that helps these melioidosis-causing bacteria to survive in the hostile, acidic environment of phagosomes. © 2017 The Authors. Published under the terms of the CC BY 4.0 license.

Comprehensive comparison of two protein family of P-ATPases (13A1 and 13A3) in insects.

Science.gov (United States)

Seddigh, Samin

2017-06-01

The P-type ATPases (P-ATPases) are present in all living cells where they mediate ion transport across membranes on the expense of ATP hydrolysis. Different ions which are transported by these pumps are protons like calcium, sodium, potassium, and heavy metals such as manganese, iron, copper, and zinc. Maintenance of the proper gradients for essential ions across cellular membranes makes P-ATPases crucial for cell survival. In this study, characterization of two families of P-ATPases including P-ATPase 13A1 and P-ATPase 13A3 protein was compared in two different insect species from different orders. According to the conserved motifs found with MEME, nine motifs were shared by insects of 13A1 family but eight in 13A3 family. Seven different insect species from 13A1 and five samples from 13A3 family were selected as the representative samples for functional and structural analyses. The structural and functional analyses were performed with ProtParam, SOPMA, SignalP 4.1, TMHMM 2.0, ProtScale and ProDom tools in the ExPASy database. The tertiary structure of Bombus terrestris as a sample of each family of insects were predicted by the Phyre2 and TM-score servers and their similarities were verified by SuperPose server. The tertiary structures were predicted via the "c3b9bA" model (PDB Accession Code: 3B9B) in P-ATPase 13A1 family and "c2zxeA" model (PDB Accession Code: 2ZXE) in P-ATPase 13A3 family. A phylogenetic tree was constructed with MEGA 6.06 software using the Neighbor-joining method. According to the results, there was a high identity of P-ATPase families so that they should be derived from a common ancestor however they belonged to separate groups. In protein-protein interaction analysis by STRING 10.0, six common enriched pathways of KEGG were identified in B. terrestris in both families. The obtained data provide a background for bioinformatic studies of the function and evolution of other insects and organisms. Copyright © 2017 Elsevier Ltd. All rights

Role of post-translational modifications at the β-subunit ectodomain in complex association with a promiscuous plant P4-ATPase.

Science.gov (United States)

Costa, Sara R; Marek, Magdalena; Axelsen, Kristian B; Theorin, Lisa; Pomorski, Thomas G; López-Marqués, Rosa L

2016-06-01

P-type ATPases of subfamily IV (P4-ATPases) constitute a major group of phospholipid flippases that form heteromeric complexes with members of the Cdc50 (cell division control 50) protein family. Some P4-ATPases interact specifically with only one β-subunit isoform, whereas others are promiscuous and can interact with several isoforms. In the present study, we used a site-directed mutagenesis approach to assess the role of post-translational modifications at the plant ALIS5 β-subunit ectodomain in the functionality of the promiscuous plant P4-ATPase ALA2. We identified two N-glycosylated residues, Asn(181) and Asn(231) Whereas mutation of Asn(231) seems to have a small effect on P4-ATPase complex formation, mutation of evolutionarily conserved Asn(181) disrupts interaction between the two subunits. Of the four cysteine residues located in the ALIS5 ectodomain, mutation of Cys(86) and Cys(107) compromises complex association, but the mutant β-subunits still promote complex trafficking and activity to some extent. In contrast, disruption of a conserved disulfide bond between Cys(158) and Cys(172) has no effect on the P4-ATPase complex. Our results demonstrate that post-translational modifications in the β-subunit have different functional roles in different organisms, which may be related to the promiscuity of the P4-ATPase. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

Evidence of two different Na/sup +/-dependent (/sup 3/H)-ouabain binding sites of a Na/sup +/-K/sup +/-ATPase of guinea-pig hearts

Energy Technology Data Exchange (ETDEWEB)

Fricke, U; Klaus, W [Koeln Univ. (Germany, F.R.)

1977-11-01

The influence of various Na/sup +/ concentrations on (/sup 3/H)-ouabain binding was studied in experiments on a microsomal Na/sup +/-K/sup +/-adenosine triphosphatase (ATPase) from guinea-pig hearts. The ATP-independent cardiac glycoside binding was not influenced by increasing Na/sup +/ concentrations. However, a good correlation was found between the ATP-dependent (/sup 3/H)-ouabain binding and Na/sup +/ concentration. A more detailed analysis of these results revealed two distinct processes involved in this interaction: one ouabain binding process was activated at rather low Na/sup +/ concentrations, (Ksub(0.5) = 4.5 mM); this type of (/sup 3/H)-ouabain binding was strongly correlated to the Na/sup +/ concentration necessary for half maximum phosphorylation (Ksub(0.5) = 1 mM). The other ouabain binding process was predominant at high Na/sup +/ concentrations (Ksub(0.5 = 69 mM). On the basis of the commonly accepted ATPase reaction cycle a model for the interaction of cardiac glycosides with the Na/sup +/-K/sup +/-ATPase is proposed, assuming two different binding sites for cardiac glycosides (E/sub 2/ -P and Esub(l) -P) and involving a translocation of these drugs from an outer to an inner compartment of the cell membrane.

Host and Pathogen Copper-Transporting P-Type ATPases Function Antagonistically during Salmonella Infection.

Science.gov (United States)

Ladomersky, Erik; Khan, Aslam; Shanbhag, Vinit; Cavet, Jennifer S; Chan, Jefferson; Weisman, Gary A; Petris, Michael J

2017-09-01

Copper is an essential yet potentially toxic trace element that is required by all aerobic organisms. A key regulator of copper homeostasis in mammalian cells is the copper-transporting P-type ATPase ATP7A, which mediates copper transport from the cytoplasm into the secretory pathway, as well as copper export across the plasma membrane. Previous studies have shown that ATP7A-dependent copper transport is required for killing phagocytosed Escherichia coli in a cultured macrophage cell line. In this investigation, we expanded on these studies by generating Atp7a LysMcre mice, in which the Atp7a gene was specifically deleted in cells of the myeloid lineage, including macrophages. Primary macrophages isolated from Atp7a LysMcre mice exhibit decreased copper transport into phagosomal compartments and a reduced ability to kill Salmonella enterica serovar Typhimurium compared to that of macrophages isolated from wild-type mice. The Atp7a LysMcre mice were also more susceptible to systemic infection by S Typhimurium than wild-type mice. Deletion of the S Typhimurium copper exporters, CopA and GolT, was found to decrease infection in wild-type mice but not in the Atp7a LysMcre mice. These studies suggest that ATP7A-dependent copper transport into the phagosome mediates host defense against S Typhimurium, which is counteracted by copper export from the bacteria via CopA and GolT. These findings reveal unique and opposing functions for copper transporters of the host and pathogen during infection. Copyright © 2017 American Society for Microbiology.

A Tonoplast P3B-ATPase Mediates Fusion of Two Types of Vacuoles in Petal Cells

Directory of Open Access Journals (Sweden)

Marianna Faraco

2017-06-01

Full Text Available It is known that plant cells can contain multiple distinct vacuoles; however, the abundance of multivacuolar cells and the mechanisms underlying vacuolar differentiation and communication among different types of vacuoles remain unknown. PH1 and PH5 are tonoplast P-ATPases that form a heteromeric pump that hyper-acidifies the central vacuole (CV of epidermal cells in petunia petals. Here, we show that the sorting of this pump and other vacuolar proteins to the CV involves transit through small vacuoles: vacuolinos. Vacuolino formation is controlled by transcription factors regulating pigment synthesis and transcription of PH1 and PH5. Trafficking of proteins from vacuolinos to the central vacuole is impaired by misexpression of vacuolar SNAREs as well as mutants for the PH1 component of the PH1-PH5 pump. The finding that PH1-PH5 and these SNAREs interact strongly suggests that structural tonoplast proteins can act as tethering factors in the recognition of different vacuolar types.

Plant P4-ATPases: lipid translocators with a role in membrane traficking

DEFF Research Database (Denmark)

Lopez Marques, Rosa Laura

a large family of membrane proteins involved in pumping different physiologically-relevant substrates across biological membranes [4]. The members of the P4 subfamily (also known as flippases) catalyze the energy-driven translocation of lipids necessary for establishing transbilayer lipid asymmetry [5......], a feature necessary for correct functioning of the cells [6,7]. Deletion of one or more P4-ATPase genes causes defects in vesicle budding in various organisms [8-10] and some members of the yeast family have been shown to interact with the vesiculation machinery [11,12]. Thus, unraveling the key features...... of P4-ATPase functioning is crucial to understand the mechanisms underlying the whole secretory and endocytic pathways. In the model plant Arabidopsis, 12 members of the P4-ATPase family have been described (ALA1-ALA12, for Aminophospholipid ATPase) [4]. In the past years, we have characterized several...

Atrial Na,K-ATPase increase and potassium dysregulation accentuate the risk of postoperative atrial fibrillation

DEFF Research Database (Denmark)

Tran, Cao Thach; Schmidt, Thomas Andersen; Christensen, John Brochorst

2009-01-01

BACKGROUND: Postoperative atrial fibrillation is a common complication to cardiac surgery. Na,K-ATPase is of major importance for the resting membrane potential and action potential. The purpose of the present study was to evaluate the importance of Na,K-ATPase concentrations in human atrial...... biopsies and plasma potassium concentrations for the development of atrial fibrillation. METHODS: Atrial myocardial biopsies were obtained from 67 patients undergoing open chest cardiac surgery. Na,K-ATPase was quantified using vanadate-facilitated 3H-ouabain binding. Plasma potassium concentration....../g wet weight (n = 33), p = 0.03]. Also with multivariable analysis, 3H-ouabain-binding site concentration was significantly associated with the development of atrial fibrillation. High increase in plasma potassium concentration during the perioperative period and surgery was associated...

Towards defining the substrate of orphan P5A-ATPases

DEFF Research Database (Denmark)

Sørensen, Danny Mollerup; Holen, Henrik Waldal; Holemans, Tine

2015-01-01

leads to broad and unspecific phenotypes related to the impairment of basic ER functions such as protein folding and processing. Genetic interactions in Saccharomyces cerevisiae point to a role of the endogenous P5A-ATPase Spf1p in separation of charges in the ER, in sterol metabolism, and in insertion...... significance Identification of the substrate of P5A-ATPases would throw light on an important general process in the ER that is still not fully understood. This article is part of a Special Issue entitled Structural biochemistry and biophysics of membrane proteins....

V-ATPase-dependent luminal acidification is required for endocytic recycling of a yeast cell wall stress sensor, Wsc1p

Energy Technology Data Exchange (ETDEWEB)

Ueno, Kazuma; Saito, Mayu; Nagashima, Makiko; Kojima, Ai; Nishinoaki, Show [Department of Biological Science and Technology, Tokyo University of Science, Niijuku 6-3-1, Katsushika-ku, Tokyo 125-8585 (Japan); Toshima, Junko Y., E-mail: yama_jun@aoni.waseda.jp [Faculty of Science and Engineering, Waseda University, Wakamatsu-cho 2-2, Shinjuku-ku, Tokyo 162-8480 (Japan); Research Center for RNA Science, RIST, Tokyo University of Science, Niijuku 6-3-1, Katsushika-ku, Tokyo 125-8585 (Japan); Toshima, Jiro, E-mail: jtosiscb@rs.noda.tus.ac.jp [Department of Biological Science and Technology, Tokyo University of Science, Niijuku 6-3-1, Katsushika-ku, Tokyo 125-8585 (Japan); Research Center for RNA Science, RIST, Tokyo University of Science, Niijuku 6-3-1, Katsushika-ku, Tokyo 125-8585 (Japan)

2014-01-10

Highlights: •A targeted genome screen identified 5 gene groups affecting Wsc1p recycling. •V-ATPase-dependent luminal acidification is required for Wsc1p recycling. •Activity of V-ATPase might be required for cargo recognition by the retromer complex. -- Abstract: Wsc1p is a major cell wall sensor protein localized at the polarized cell surface. The localization of Wsc1p is maintained by endocytosis and recycling from endosomes back to the cell surface, but changes to the vacuole when cells are subjected to heat stress. Exploiting this unique property of Wsc1p, we screened for yeast single-gene deletion mutants exhibiting defects in Wsc1p trafficking. By expressing 3GFP-tagged Wsc1p in mutants with deleted genes whose function is related to intracellular trafficking, we identified 5 gene groups affecting Wsc1p trafficking, impaired respectively in endocytic internalization, multivesicular body sorting, the GARP complex, endosomal maturation/vacuolar fusion, and V-ATPase. Interestingly, deletion of the VPH1 gene, encoding the V{sub o} subunit of vacuolar-type H{sup +}-ATPase (V-ATPase), led to mis-localization of Wsc1p from the plasma membrane to the vacuole. In addition, disruption of other V-ATPase subunits (vma mutants) also caused defects of Wsc1p trafficking and vacuolar acidification similar to those seen in the vph1Δ mutant. Moreover, we found that deletion of the VPS26 gene, encoding a subunit of the retromer complex, also caused a defect in Wsc1p recycling and mis-localization of Wsc1p to the vacuole. These findings clarified the previously unidentified Wsc1p recycling pathway and requirement of V-ATPase-dependent luminal acidification for Wsc1p recycling.

V-ATPase-dependent luminal acidification is required for endocytic recycling of a yeast cell wall stress sensor, Wsc1p

International Nuclear Information System (INIS)

Ueno, Kazuma; Saito, Mayu; Nagashima, Makiko; Kojima, Ai; Nishinoaki, Show; Toshima, Junko Y.; Toshima, Jiro

2014-01-01

Highlights: •A targeted genome screen identified 5 gene groups affecting Wsc1p recycling. •V-ATPase-dependent luminal acidification is required for Wsc1p recycling. •Activity of V-ATPase might be required for cargo recognition by the retromer complex. -- Abstract: Wsc1p is a major cell wall sensor protein localized at the polarized cell surface. The localization of Wsc1p is maintained by endocytosis and recycling from endosomes back to the cell surface, but changes to the vacuole when cells are subjected to heat stress. Exploiting this unique property of Wsc1p, we screened for yeast single-gene deletion mutants exhibiting defects in Wsc1p trafficking. By expressing 3GFP-tagged Wsc1p in mutants with deleted genes whose function is related to intracellular trafficking, we identified 5 gene groups affecting Wsc1p trafficking, impaired respectively in endocytic internalization, multivesicular body sorting, the GARP complex, endosomal maturation/vacuolar fusion, and V-ATPase. Interestingly, deletion of the VPH1 gene, encoding the V o subunit of vacuolar-type H + -ATPase (V-ATPase), led to mis-localization of Wsc1p from the plasma membrane to the vacuole. In addition, disruption of other V-ATPase subunits (vma mutants) also caused defects of Wsc1p trafficking and vacuolar acidification similar to those seen in the vph1Δ mutant. Moreover, we found that deletion of the VPS26 gene, encoding a subunit of the retromer complex, also caused a defect in Wsc1p recycling and mis-localization of Wsc1p to the vacuole. These findings clarified the previously unidentified Wsc1p recycling pathway and requirement of V-ATPase-dependent luminal acidification for Wsc1p recycling

Plasmalemmal V-H+-ATPases regulate intracellular pH in human lung microvascular endothelial cells

International Nuclear Information System (INIS)

Rojas, Jose D.; Sennoune, Souad R.; Maiti, Debasish; Martinez, Gloria M.; Bakunts, Karina; Wesson, Donald E.; Martinez-Zaguilan, Raul

2004-01-01

The lung endothelium layer is exposed to continuous CO 2 transit which exposes the endothelium to a substantial acid load that could be detrimental to cell function. The Na + /H + exchanger and HCO 3 - -dependent H + -transporting mechanisms regulate intracellular pH (pH cyt ) in most cells. Cells that cope with high acid loads might require additional primary energy-dependent mechanisms. V-H + -ATPases localized at the plasma membranes (pmV-ATPases) have emerged as a novel pH regulatory system. We hypothesized that human lung microvascular endothelial (HLMVE) cells use pmV-ATPases, in addition to Na + /H + exchanger and HCO 3 - -based H + -transporting mechanisms, to maintain pH cyt homeostasis. Immunocytochemical studies revealed V-H + -ATPase at the plasma membrane, in addition to the predicted distribution in vacuolar compartments. Acid-loaded HLMVE cells exhibited proton fluxes in the absence of Na + and HCO 3 - that were similar to those observed in the presence of either Na + , or Na + and HCO 3 - . The Na + - and HCO 3 - -independent pH cyt recovery was inhibited by bafilomycin A 1 , a V-H + -ATPase inhibitor. These studies show a Na + - and HCO 3 - -independent pH cyt regulatory mechanism in HLMVE cells that is mediated by pmV-ATPases

Zebrafish chemical screening reveals the impairment of dopaminergic neuronal survival by cardiac glycosides.

Directory of Open Access Journals (Sweden)

Yaping Sun

Full Text Available Parkinson's disease is a neurodegenerative disorder characterized by the prominent degeneration of dopaminergic (DA neurons among other cell types. Here we report a first chemical screen of over 5,000 compounds in zebrafish, aimed at identifying small molecule modulators of DA neuron development or survival. We find that Neriifolin, a member of the cardiac glycoside family of compounds, impairs survival but not differentiation of both zebrafish and mammalian DA neurons. Cardiac glycosides are inhibitors of Na(+/K(+ ATPase activity and widely used for treating heart disorders. Our data suggest that Neriifolin impairs DA neuronal survival by targeting the neuronal enriched Na(+/K(+ ATPase α3 subunit (ATP1A3. Modulation of ionic homeostasis, knockdown of p53, or treatment with antioxidants protects DA neurons from Neriifolin-induced death. These results reveal a previously unknown effect of cardiac glycosides on DA neuronal survival and suggest that it is mediated through ATP1A3 inhibition, oxidative stress, and p53. They also elucidate potential approaches for counteracting the neurotoxicity of this valuable class of medications.

Overproduction of PIB-Type ATPases

DEFF Research Database (Denmark)

Liu, Xiangyu; Sitsel, Oleg; Wang, Kaituo

2016-01-01

Understanding of the functions and mechanisms of fundamental processes in the cell requires structural information. Structural studies of membrane proteins typically necessitate large amounts of purified and preferably homogenous target protein. Here, we describe a rapid overproduction and purifi...... and purification strategy of a bacterial PIB-type ATPase for isolation of milligrams of target protein per liter Escherichia coli cell culture, with a final quality of the sample which is sufficient for generating high-resolution crystals....

Active ingredients in Chinese medicines promoting blood circulation as Na+/K+ -ATPase inhibitors.

Science.gov (United States)

Chen, Ronald J Y; Jinn, Tzyy-rong; Chen, Yi-ching; Chung, Tse-yu; Yang, Wei-hung; Tzen, Jason T C

2011-02-01

The positive inotropic effect of cardiac glycosides lies in their reversible inhibition on the membrane-bound Na(+)/K(+)-ATPase in human myocardium. Steroid-like compounds containing a core structure similar to cardiac glycosides are found in many Chinese medicines conventionally used for promoting blood circulation. Some of them are demonstrated to be Na(+)/K(+)-ATPase inhibitors and thus putatively responsible for their therapeutic effects via the same molecular mechanism as cardiac glycosides. On the other hand, magnesium lithospermate B of danshen is also proposed to exert its cardiac therapeutic effect by effectively inhibiting Na(+)/K(+)-ATPase. Theoretical modeling suggests that the number of hydrogen bonds and the strength of hydrophobic interaction between the effective ingredients of various medicines and residues around the binding pocket of Na(+)/K(+)-ATPase are crucial for the inhibitory potency of these active ingredients. Ginsenosides, the active ingredients in ginseng and sanqi, substantially inhibit Na(+)/K(+)-ATPase when sugar moieties are attached only to the C-3 position of their steroid-like structure, equivalent to the sugar position in cardiac glycosides. Their inhibitory potency is abolished, however, when sugar moieties are linked to C-6 or C-20 position of the steroid nucleus; presumably, these sugar attachments lead to steric hindrance for the entrance of ginsenosides into the binding pocket of Na(+)/K(+)-ATPase. Neuroprotective effects of cardiac glycosides, several steroid-like compounds, and magnesium lithospermate B against ischemic stroke have been accordingly observed in a cortical brain slice-based assay model, and cumulative data support that effective inhibitors of Na(+)/K(+)-ATPase in the brain could be potential drugs for the treatment of ischemic stroke.

The AAA+ ATPase p97, a cellular multitool.

Science.gov (United States)

Stach, Lasse; Freemont, Paul S

2017-08-17

The AAA+ (ATPases associated with diverse cellular activities) ATPase p97 is essential to a wide range of cellular functions, including endoplasmic reticulum-associated degradation, membrane fusion, NF-κB (nuclear factor kappa-light-chain-enhancer of activated B cells) activation and chromatin-associated processes, which are regulated by ubiquitination. p97 acts downstream from ubiquitin signaling events and utilizes the energy from ATP hydrolysis to extract its substrate proteins from cellular structures or multiprotein complexes. A multitude of p97 cofactors have evolved which are essential to p97 function. Ubiquitin-interacting domains and p97-binding domains combine to form bi-functional cofactors, whose complexes with p97 enable the enzyme to interact with a wide range of ubiquitinated substrates. A set of mutations in p97 have been shown to cause the multisystem proteinopathy inclusion body myopathy associated with Paget's disease of bone and frontotemporal dementia. In addition, p97 inhibition has been identified as a promising approach to provoke proteotoxic stress in tumors. In this review, we will describe the cellular processes governed by p97, how the cofactors interact with both p97 and its ubiquitinated substrates, p97 enzymology and the current status in developing p97 inhibitors for cancer therapy. © 2017 The Author(s).

Intact long-type DupA protein in Helicobacter pylori is an ATPase involved in multifunctional biological activities.

Science.gov (United States)

Wang, Ming-yi; Chen, Cheng; Shao, Chen; Wang, Shao-bo; Wang, Ai-chu; Yang, Ya-chao; Yuan, Xiao-yan; Shao, Shi-he

2015-04-01

The function of intact long-type DupA protein in Helicobacter pylori was analyzed using immunoblotting and molecular biology techniques in the study. After cloning, expression and purification, ATPase activity of DupA protein was detected. Antibody was produced for localization and interaction proteins analysis. The dupA-deleted mutant was generated for adhesion and CagA protein translocation assay, susceptibility to different pH, IL-8 secretion assay, cytotoxicity to MKN-45 cells and proteins-involved apoptosis analysis. DupA protein exhibited an ATPase activity (129.5±17.8 U/mgprot) and located in bacterial membrane, while it did not involve the adhesion and CagA protein delivery of H. pylori. DupA protein involved the urease secretion as the interaction proteins. The wild type strain had a stronger growth in low pH than the dupA-deleted mutant (p DupA protein located in membrane as ATPase is a true virulence factor associated with duodenal ulcer development involving the IL-8 induction and urease secretion, while it inhibits gastric cancer cell growth in vitro by activating the mitochondria-mediated apoptotic pathway. Copyright © 2015 Elsevier Ltd. All rights reserved.

Two types of essential carboxyl groups in Rhodospirillum rubrum proton ATPase

International Nuclear Information System (INIS)

Ceccarelli, E.; Vallejos, R.H.

1983-01-01

Two different types of essential carboxyl groups were detected in the extrinsic component of the proton ATPase of Rhodospirillum rubrum. Chemical modification of R. rubrum chromatophores or its solubilized ATPase by Woodward's reagent K resulted in inactivation of photophosphorylating and ATPase activities. The apparent order of reaction was nearly 1 with respect to reagent concentration and similar K1 were obtained for the soluble and membrane-bound ATPases suggesting that inactivation was associated with modification of one essential carboxyl group located in the soluble component of the proton ATPase. Inactivation was prevented by adenine nucleotides but not by divalent cations. Dicyclohexylcarbodiimide completely inhibited the solubilized ATPase with a K1 of 5.2 mM and a K2 of 0.81 min-1. Mg2+ afforded nearly complete protection with a Kd of 2.8 mM. Two moles of [14C]dicyclohexylcarbodiimide were incorporated per mole of enzyme for complete inactivation but in the presence of 30 mM MgCl2 only one mole was incorporated and there was no inhibition. The labeling was recovered mostly from the beta subunit. The incorporation of the labeled reagent into the ATPase was not prevented by previous modification with Woodward's reagent K. It is concluded that both reagents modified two different essential carboxyl groups in the soluble ATPase from R. rubrum

The amino-terminal 200 amino acids of the plasma membrane Na+,K+-ATPase alpha subunit confer ouabain sensitivity on the sarcoplasmic reticulum Ca(2+)-ATPase.

OpenAIRE

Ishii, T; Takeyasu, K

1993-01-01

Cardiac glycosides such as G-strophanthin (ouabain) bind to and inhibit the plasma membrane Na+,K(+)-ATPase but not the sarcoplasmic reticulum (SR) Ca(2+)-ATPase, whereas thapsigargin specifically blocks the SR Ca(2+)-ATPase. The chimera [n/c]CC, in which the amino-terminal amino acids Met1 to Asp162 of the SR Ca(2+)-ATPase (SERCA1) were replaced with the corresponding portion of the Na+,K(+)-ATPase alpha 1 subunit (Met1 to Asp200), retained thapsigargin- and Ca(2+)-sensitive ATPase activity,...

A structural overview of the plasma membrane Na+,K+-ATPase and H+-ATPase ion pumps

DEFF Research Database (Denmark)

Morth, Jens Preben; Pedersen, Bjørn Panella; Buch-Pedersen, Morten Jeppe

2011-01-01

transport systems that are responsible for uptake and extrusion of metabolites and other ions. The ion gradients are also both directly and indirectly used to control pH homeostasis and to regulate cell volume. The plasma membrane H(+)-ATPase maintains a proton gradient in plants and fungi and the Na......Plasma membrane ATPases are primary active transporters of cations that maintain steep concentration gradients. The ion gradients and membrane potentials derived from them form the basis for a range of essential cellular processes, in particular Na(+)-dependent and proton-dependent secondary......(+),K(+)-ATPase maintains a Na(+) and K(+) gradient in animal cells. Structural information provides insight into the function of these two distinct but related P-type pumps....

A structural overview of the plasma membrane Na+,K+-ATPase and H+-ATPase ion pumps

DEFF Research Database (Denmark)

Morth, Jens Preben; Pedersen, Bjørn Panella; Buch-Pedersen, Morten Jeppe

2011-01-01

transport systems that are responsible for uptake and extrusion of metabolites and other ions. The ion gradients are also both directly and indirectly used to control pH homeostasis and to regulate cell volume. The plasma membrane H(+)-ATPase maintains a proton gradient in plants and fungi and the Na(+),K(+)-ATPase...... maintains a Na(+) and K(+) gradient in animal cells. Structural information provides insight into the function of these two distinct but related P-type pumps.......Plasma membrane ATPases are primary active transporters of cations that maintain steep concentration gradients. The ion gradients and membrane potentials derived from them form the basis for a range of essential cellular processes, in particular Na(+)-dependent and proton-dependent secondary...

Reconstruction of the complete ouabain-binding pocket of Na,K-ATPase in gastric H,K-ATPase by substitution of only seven amino acids.

Science.gov (United States)

Qiu, Li Yan; Krieger, Elmar; Schaftenaar, Gijs; Swarts, Herman G P; Willems, Peter H G M; De Pont, Jan Joep H H M; Koenderink, Jan B

2005-09-16

Although cardiac glycosides have been used as drugs for more than 2 centuries and their primary target, the sodium pump (Na,K-ATPase), has already been known for 4 decades, their exact binding site is still elusive. In our efforts to define the molecular basis of digitalis glycosides binding we started from the fact that a closely related enzyme, the gastric H,K-ATPase, does not bind glycosides like ouabain. Previously, we showed that a chimera of these two enzymes, in which only the M3-M4 and M5-M6 hairpins were of Na,K-ATPase, bound ouabain with high affinity (Koenderink, J. B., Hermsen, H. P. H., Swarts, H. G. P., Willems, P. H. G. M., and De Pont, J. J. H. H. M. (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 11209-11214). We also demonstrated that only three amino acids (Phe(783), Thr(797), and Asp(804)) present in the M5-M6 hairpin of Na,K-ATPase were sufficient to confer high affinity ouabain binding to a chimera which contained in addition the M3-M4 hairpin of Na,K-ATPase (Qiu, L. Y., Koenderink, J. B., Swarts, H. G., Willems, P. H., and De Pont, J. J. H. H. M. (2003) J. Biol. Chem. 278, 47240-47244). To further pinpoint the ouabain-binding site here we used a chimera-based loss-of-function strategy and identified four amino acids (Glu(312), Val(314), Ile(315), Gly(319)), all present in M4, as being important for ouabain binding. In a final gain-of-function study we showed that a gastric H,K-ATPase that contained Glu(312), Val(314), Ile(315), Gly(319), Phe(783), Thr(797), and Asp(804) of Na,K-ATPase bound ouabain with the same affinity as the native enzyme. Based on the E(2)P crystal structure of Ca(2+)-ATPase we constructed a homology model for the ouabain-binding site of Na,K-ATPase involving all seven amino acids as well as several earlier postulated amino acids.

Glycolytic control of vacuolar-type ATPase activity: A mechanism to regulate influenza viral infection

Energy Technology Data Exchange (ETDEWEB)

Kohio, Hinissan P.; Adamson, Amy L., E-mail: aladamso@uncg.edu

2013-09-15

As new influenza virus strains emerge, finding new mechanisms to control infection is imperative. In this study, we found that we could control influenza infection of mammalian cells by altering the level of glucose given to cells. Higher glucose concentrations induced a dose-specific increase in influenza infection. Linking influenza virus infection with glycolysis, we found that viral replication was significantly reduced after cells were treated with glycolytic inhibitors. Addition of extracellular ATP after glycolytic inhibition restored influenza infection. We also determined that higher levels of glucose promoted the assembly of the vacuolar-type ATPase within cells, and increased vacuolar-type ATPase proton-transport activity. The increase of viral infection via high glucose levels could be reversed by inhibition of the proton pump, linking glucose metabolism, vacuolar-type ATPase activity, and influenza viral infection. Taken together, we propose that altering glucose metabolism may be a potential new approach to inhibit influenza viral infection. - Highlights: • Increased glucose levels increase Influenza A viral infection of MDCK cells. • Inhibition of the glycolytic enzyme hexokinase inhibited Influenza A viral infection. • Inhibition of hexokinase induced disassembly the V-ATPase. • Disassembly of the V-ATPase and Influenza A infection was bypassed with ATP. • The state of V-ATPase assembly correlated with Influenza A infection of cells.

Glycolytic control of vacuolar-type ATPase activity: A mechanism to regulate influenza viral infection

International Nuclear Information System (INIS)

Kohio, Hinissan P.; Adamson, Amy L.

2013-01-01

As new influenza virus strains emerge, finding new mechanisms to control infection is imperative. In this study, we found that we could control influenza infection of mammalian cells by altering the level of glucose given to cells. Higher glucose concentrations induced a dose-specific increase in influenza infection. Linking influenza virus infection with glycolysis, we found that viral replication was significantly reduced after cells were treated with glycolytic inhibitors. Addition of extracellular ATP after glycolytic inhibition restored influenza infection. We also determined that higher levels of glucose promoted the assembly of the vacuolar-type ATPase within cells, and increased vacuolar-type ATPase proton-transport activity. The increase of viral infection via high glucose levels could be reversed by inhibition of the proton pump, linking glucose metabolism, vacuolar-type ATPase activity, and influenza viral infection. Taken together, we propose that altering glucose metabolism may be a potential new approach to inhibit influenza viral infection. - Highlights: • Increased glucose levels increase Influenza A viral infection of MDCK cells. • Inhibition of the glycolytic enzyme hexokinase inhibited Influenza A viral infection. • Inhibition of hexokinase induced disassembly the V-ATPase. • Disassembly of the V-ATPase and Influenza A infection was bypassed with ATP. • The state of V-ATPase assembly correlated with Influenza A infection of cells

Regulation of the Plasma Membrane H+-ATPase

DEFF Research Database (Denmark)

Falhof, Janus

The plasma membrane (PM) H+-ATPase is responsible for generating the electrochemical gradientthat drives the secondary transport of nutrients across the cellular membrane. It belongs to a familyof cation and lipid transporters that are vital to many organisms. PM H+-ATPases are Type P3AATPases...

Cryo-EM studies of the structure and dynamics of vacuolar-type ATPases

Science.gov (United States)

Mazhab-Jafari, Mohammad T.; Rubinstein, John L.

2016-01-01

Electron cryomicroscopy (cryo-EM) has significantly advanced our understanding of molecular structure in biology. Recent innovations in both hardware and software have made cryo-EM a viable alternative for targets that are not amenable to x-ray crystallography or nuclear magnetic resonance (NMR) spectroscopy. Cryo-EM has even become the method of choice in some situations where x-ray crystallography and NMR spectroscopy are possible but where cryo-EM can determine structures at higher resolution or with less time or effort. Rotary adenosine triphosphatases (ATPases) are crucial to the maintenance of cellular homeostasis. These enzymes couple the synthesis or hydrolysis of adenosine triphosphate to the use or production of a transmembrane electrochemical ion gradient, respectively. However, the membrane-embedded nature and conformational heterogeneity of intact rotary ATPases have prevented their high-resolution structural analysis to date. Recent application of cryo-EM methods to the different types of rotary ATPase has led to sudden advances in understanding the structure and function of these enzymes, revealing significant conformational heterogeneity and characteristic transmembrane α helices that are highly tilted with respect to the membrane. In this Review, we will discuss what has been learned recently about rotary ATPase structure and function, with a particular focus on the vacuolar-type ATPases. PMID:27532044

Structural and functional characterization of P4-ATPase lipid flippases

DEFF Research Database (Denmark)

Ulstrup, Jakob

2018-01-01

to its much larger substrate and how the mechanism allowing the transport unfolds. This is one of the central questions in the field known as the “giant substrate problem”. To this date, no structural information of P4-ATPases is available. The focus of this thesis is divided into two projects, both...... focusing on P4-ATPases from the yeast organism Saccharomyces cerevisiae: I. The structural characterization of the flippase Drs2p in complex with its auxiliary subunit Cdc50p. II. Establishing a protocol for obtaining a homogenous sample of the flippase Neo1p suitable for biochemical characterization...... and substrate identification. Part I was performed using X-ray crystallography and single-particle electron microscopy as the main methods. A 3D envelope was obtained by cryo-EM extending to a resolution of 4.4 Å. This envelope reveals the first structural insight of the conformational organization of the Drs2p...

Molecular mechanism of bacterial Hsp90 pH-dependent ATPase activity.

Science.gov (United States)

Jin, Yi; Hoxie, Reyal S; Street, Timothy O

2017-06-01

Hsp90 is a dimeric molecular chaperone that undergoes an essential and highly regulated open-to-closed-to-open conformational cycle upon ATP binding and hydrolysis. Although it has been established that a large energy barrier to closure is responsible for Hsp90's low ATP hydrolysis rate, the specific molecular contacts that create this energy barrier are not known. Here we discover that bacterial Hsp90 (HtpG) has a pH-dependent ATPase activity that is unique among other Hsp90 homologs. The underlying mechanism is a conformation-specific electrostatic interaction between a single histidine, H255, and bound ATP. H255 stabilizes ATP only while HtpG adopts a catalytically inactive open configuration, resulting in a striking anti-correlation between nucleotide binding affinity and chaperone activity over a wide range of pH. Linkage analysis reveals that the H255-ATP salt bridge contributes 1.5 kcal/mol to the energy barrier of closure. This energetic contribution is structurally asymmetric, whereby only one H255-ATP salt-bridge per dimer of HtpG controls ATPase activation. We find that a similar electrostatic mechanism regulates the ATPase of the endoplasmic reticulum Hsp90, and that pH-dependent activity can be engineered into eukaryotic cytosolic Hsp90. These results reveal site-specific energetic information about an evolutionarily conserved conformational landscape that controls Hsp90 ATPase activity. © 2017 The Protein Society.

Cryo-EM of the pathogenic VCP variant R155P reveals long-range conformational changes in the D2 ATPase ring

Energy Technology Data Exchange (ETDEWEB)

Mountassif, Driss; Fabre, Lucien [Department of Anatomy and Cell Biology, McGill University, Groupe de recherche axé sur la structure des protéines (GRASP), Groupe d' Étude des Proteines Membranaires (GÉPROM), 3640 University Street, Montreal H3A 0C7 (Canada); Zaid, Younes [Department of Anatomy and Cell Biology, McGill University, Groupe de recherche axé sur la structure des protéines (GRASP), Groupe d' Étude des Proteines Membranaires (GÉPROM), 3640 University Street, Montreal H3A 0C7 (Canada); Current address: Laboratory of Thrombosis and Hemostasis, Montreal Heart Institute, Montreal, Quebec (Canada); Halawani, Dalia [Department of Anatomy and Cell Biology, McGill University, Groupe de recherche axé sur la structure des protéines (GRASP), Groupe d' Étude des Proteines Membranaires (GÉPROM), 3640 University Street, Montreal H3A 0C7 (Canada); Current address: Department of Cell Biology, Lerner Research Institute, 9500 Euclid Avenue NC10, Cleveland, OH 44195 (United States); Rouiller, Isabelle, E-mail: isabelle.rouiller@mcgill.ca [Department of Anatomy and Cell Biology, McGill University, Groupe de recherche axé sur la structure des protéines (GRASP), Groupe d' Étude des Proteines Membranaires (GÉPROM), 3640 University Street, Montreal H3A 0C7 (Canada)

2015-12-25

Single amino acid mutations in valosin containing protein (VCP/p97), a highly conserved member of the ATPases associated with diverse cellular activities (AAA) family of ATPases has been linked to a severe degenerative disease affecting brain, muscle and bone tissue. Previous studies have demonstrated the role of VCP mutations in altering the ATPase activity of the D2 ring; however the structural consequences of these mutations remain unclear. In this study, we report the three-dimensional (3D) map of the pathogenic VCP variant, R155P, as revealed by single-particle Cryo-Electron Microscopy (EM) analysis at 14 Å resolution. We show that the N-terminal R155P mutation induces a large structural reorganisation of the D2 ATPase ring. Results from docking studies using crystal structure data of available wild-type VCP in the EM density maps indicate that the major difference is localized at the interface between two protomers within the D2 ring. Consistent with a conformational change, the VCP R155P variant shifted the isoelectric point of the protein and reduced its interaction with its well-characterized cofactor, nuclear protein localization-4 (Npl4). Together, our results demonstrate that a single amino acid substitution in the N-terminal domain can relay long-range conformational changes to the distal D2 ATPase ring. Our results provide the first structural clues of how VCP mutations may influence the activity and function of the D2 ATPase ring. - Highlights: • p97{sub R155P} and p97{sub A232E} decrease the ability of p97 to bind to its co-factor Npl4. • p97{sub R155P} has a different isoelectric point than that of p97{sub R95G}, p97{sub A232E} and p97{sub WT}. • Mutation R155P changes principally the conformation of the D2 ring. • Mutation R155P modifies the interface between two protomers within the D2 ring.

Cryo-EM of the pathogenic VCP variant R155P reveals long-range conformational changes in the D2 ATPase ring

International Nuclear Information System (INIS)

Mountassif, Driss; Fabre, Lucien; Zaid, Younes; Halawani, Dalia; Rouiller, Isabelle

2015-01-01

Single amino acid mutations in valosin containing protein (VCP/p97), a highly conserved member of the ATPases associated with diverse cellular activities (AAA) family of ATPases has been linked to a severe degenerative disease affecting brain, muscle and bone tissue. Previous studies have demonstrated the role of VCP mutations in altering the ATPase activity of the D2 ring; however the structural consequences of these mutations remain unclear. In this study, we report the three-dimensional (3D) map of the pathogenic VCP variant, R155P, as revealed by single-particle Cryo-Electron Microscopy (EM) analysis at 14 Å resolution. We show that the N-terminal R155P mutation induces a large structural reorganisation of the D2 ATPase ring. Results from docking studies using crystal structure data of available wild-type VCP in the EM density maps indicate that the major difference is localized at the interface between two protomers within the D2 ring. Consistent with a conformational change, the VCP R155P variant shifted the isoelectric point of the protein and reduced its interaction with its well-characterized cofactor, nuclear protein localization-4 (Npl4). Together, our results demonstrate that a single amino acid substitution in the N-terminal domain can relay long-range conformational changes to the distal D2 ATPase ring. Our results provide the first structural clues of how VCP mutations may influence the activity and function of the D2 ATPase ring. - Highlights: • p97 R155P and p97 A232E decrease the ability of p97 to bind to its co-factor Npl4. • p97 R155P has a different isoelectric point than that of p97 R95G , p97 A232E and p97 WT . • Mutation R155P changes principally the conformation of the D2 ring. • Mutation R155P modifies the interface between two protomers within the D2 ring.

Modulation and Functional Role of the Orientations of the N- and P-Domains of Cu+ -Transporting ATPase along the Ion Transport Cycle.

Science.gov (United States)

Meng, Dan; Bruschweiler-Li, Lei; Zhang, Fengli; Brüschweiler, Rafael

2015-08-18

Ion transport of different P-type ATPases is regulated similarly through the interplay of multiple protein domains. In the presence of ATP, binding of a cation to the ion binding site in the transmembrane helices leads to the phosphorylation of the P-domain, allowing ion transfer across the membrane. The details of the mechanism, however, are not clear. Here, we report the modulation of the orientation between the N- and P-domains of Cu(+)-transporting ATPase along the ion transport cycle using high-resolution nuclear magnetic resonance spectroscopy in solution. On the basis of residual dipolar coupling measurements, it is found that the interdomain orientation (relative openness) of the N- and P-domains is distinctly modulated depending on the specific state of the N- and P-domains along the ion translocation cycle. The two domains' relative position in the apo state is semiopen, whereas it becomes closed upon binding of ATP to the N-domain. After phosphorylation of the P-domain and the release of ADP, the opening, however, becomes the widest among all the states. We reason such wide opening resulting from the departure of ADP prepares the N- and P-domains to accommodate the A-domain for interaction and, hence, promote ion transport and allow dephosphorylation of the P-domain. Such wide interdomain opening is abolished when an Asn to Asp mutation is introduced into the conserved DXXK motif located in the hinge region of the N- and P-domains of Cu(+)-ATPase, suggesting the indispensible role of the N- and P-interdomain orientation during ion transportation. Our results shed new light on the structural and mechanistic details of P-type ATPase function at large.

Crystal structure of the sodium-potassium pump (Na+,K+-ATPase) with bound potassium and ouabain

OpenAIRE

Ogawa, Haruo; Shinoda, Takehiro; Cornelius, Flemming; Toyoshima, Chikashi

2009-01-01

The sodium-potassium pump (Na+,K+-ATPase) is responsible for establishing Na+ and K+ concentration gradients across the plasma membrane and therefore plays an essential role in, for instance, generating action potentials. Cardiac glycosides, prescribed for congestive heart failure for more than 2 centuries, are efficient inhibitors of this ATPase. Here we describe a crystal structure of Na+,K+-ATPase with bound ouabain, a representative cardiac glycoside, at 2.8 Å resolution in a state analog...

Cryo-EM of the pathogenic VCP variant R155P reveals long-range conformational changes in the D2 ATPase ring.

Science.gov (United States)

Mountassif, Driss; Fabre, Lucien; Zaid, Younes; Halawani, Dalia; Rouiller, Isabelle

2015-12-25

Single amino acid mutations in valosin containing protein (VCP/p97), a highly conserved member of the ATPases associated with diverse cellular activities (AAA) family of ATPases has been linked to a severe degenerative disease affecting brain, muscle and bone tissue. Previous studies have demonstrated the role of VCP mutations in altering the ATPase activity of the D2 ring; however the structural consequences of these mutations remain unclear. In this study, we report the three-dimensional (3D) map of the pathogenic VCP variant, R155P, as revealed by single-particle Cryo-Electron Microscopy (EM) analysis at 14 Å resolution. We show that the N-terminal R155P mutation induces a large structural reorganisation of the D2 ATPase ring. Results from docking studies using crystal structure data of available wild-type VCP in the EM density maps indicate that the major difference is localized at the interface between two protomers within the D2 ring. Consistent with a conformational change, the VCP R155P variant shifted the isoelectric point of the protein and reduced its interaction with its well-characterized cofactor, nuclear protein localization-4 (Npl4). Together, our results demonstrate that a single amino acid substitution in the N-terminal domain can relay long-range conformational changes to the distal D2 ATPase ring. Our results provide the first structural clues of how VCP mutations may influence the activity and function of the D2 ATPase ring. Copyright © 2015 Elsevier Inc. All rights reserved.

Structural and functional studies of a Cu+-ATPase from Legionella pneumophila

DEFF Research Database (Denmark)

Mattle, Daniel

During his studies, Daniel Mattle explored the copper(I) export mechanism of a P-type Cu+ ATPase from Legionella pneumophila – a homologue to the human Cu+ ATPases. Cu+ ATPases are responsible for the homeostatic control of the physiological relevant – but toxic – copper(I) cations. To assess...

Oxidant-NO dependent gene regulation in dogs with type I diabetes: impact on cardiac function and metabolism

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Ojaimi Caroline

2010-08-01

Full Text Available Abstract Background The mechanisms responsible for the cardiovascular mortality in type I diabetes (DM have not been defined completely. We have shown in conscious dogs with DM that: 1 baseline coronary blood flow (CBF was significantly decreased, 2 endothelium-dependent (ACh coronary vasodilation was impaired, and 3 reflex cholinergic NO-dependent coronary vasodilation was selectively depressed. The most likely mechanism responsible for the depressed reflex cholinergic NO-dependent coronary vasodilation was the decreased bioactivity of NO from the vascular endothelium. The goal of this study was to investigate changes in cardiac gene expression in a canine model of alloxan-induced type 1 diabetes. Methods Mongrel dogs were chronically instrumented and the dogs were divided into two groups: one normal and the other diabetic. In the diabetic group, the dogs were injected with alloxan monohydrate (40-60 mg/kg iv over 1 min. The global changes in cardiac gene expression in dogs with alloxan-induced diabetes were studied using Affymetrix Canine Array. Cardiac RNA was extracted from the control and DM (n = 4. Results The array data revealed that 797 genes were differentially expressed (P 2+ cycling genes (ryanodine receptor; SERCA2 Calcium ATPase, structural proteins (actin alpha. Of particular interests are genes involved in glutathione metabolism (glutathione peroxidase 1, glutathione reductase and glutathione S-transferase, which were markedly down regulated. Conclusion our findings suggest that type I diabetes might have a direct effect on the heart by impairing NO bioavailability through oxidative stress and perhaps lipid peroxidases.

Active compounds in Chinese herbs and medicinal animal products which promote blood circulation via inhibition of Na+, K+-ATPase.

Science.gov (United States)

Tzen, Jason Tc; Chen, Ronald Jy; Chung, Tse-Yu; Chen, Yi-Ching; Lin, Nan-Hei

2010-01-01

The therapeutic effect of cardiac glycosides for congestive heart failure lies in their reversible inhibition on Na+, K+-ATPase located in human myocardium. Several steroid-like compounds containing a core structure similar to cardiac glycosides have been found in many Chinese herbs and medicinal animal products conventionally used to promote blood circulation. They are putatively responsible for the therapeutic effect of those medicinal products via the same mechanism of inhibiting Na+, K+-ATPase. Inhibitory potency on Na+, K+-ATPase by ginsenosides, one of the identified steroid-like compounds, is significantly affected by sugar attachment that might cause steric hindrance of their binding to Na+, K+-ATPase. Ginsenosides with sugar moieties attached only to the C-3 position of the steroid-like structure, equivalent to the sugar position in cardiac glycosides, substantially inhibit Na+, K+-ATPase. However, their inhibitory potency is abolished when sugar moieties are linked to the C-6 or C-20 position of the steroid-like structure. In contrast, no appreciable contents of steroid-like compounds are found in danshen, a well-known Chinese herb traditionally regarded as an effective medicine promoting blood circulation. Instead, magnesium lithospermate B (MLB), the major soluble ingredient in danshen, is assumed to be responsible for the therapeutic effect by inhibiting Na+, K+-ATPase in a manner comparable to cardiac glycosides. Neuroprotective effects of cardiac glycosides, ginsenosides and MLB against ischemic stroke were accordingly observed in a cortical brain slice-based assay model. Whether the neuroprotection is also triggered by inhibition of Na+, K+-ATPase remains to be investigated. Molecular modeling suggests that cardiac glycosides, ginsenosides and MLB presumably bind to the same extracellular pocket of the Na+, K+-ATPase alpha subunit.

Structure of V-type ATPase from Clostridium fervidus by electron microscopy

NARCIS (Netherlands)

Boekema, EJ; Ubbink-Kok, T; Lolkema, JS; Brisson, A; Konings, WN

F-type and V-type ATPases couple synthesis or hydrolysis of ATP to the translocation of H+ or Na+ across biological membranes and have similarities in structure and mechanism. In both types of enzymes three main parts can be distinguished: headpiece, membrane-bound piece and stalk region. We report

Development of Classification Models for Identifying “True” P-glycoprotein (P-gp Inhibitors Through Inhibition, ATPase Activation and Monolayer Efflux Assays

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Anna Maria Bianucci

2012-06-01

Full Text Available P-glycoprotein (P-gp is an efflux pump involved in the protection of tissues of several organs by influencing xenobiotic disposition. P-gp plays a key role in multidrug resistance and in the progression of many neurodegenerative diseases. The development of new and more effective therapeutics targeting P-gp thus represents an intriguing challenge in drug discovery. P-gp inhibition may be considered as a valid approach to improve drug bioavailability as well as to overcome drug resistance to many kinds of tumours characterized by the over-expression of this protein. This study aims to develop classification models from a unique dataset of 59 compounds for which there were homogeneous experimental data on P-gp inhibition, ATPase activation and monolayer efflux. For each experiment, the dataset was split into a training and a test set comprising 39 and 20 molecules, respectively. Rational splitting was accomplished using a sphere-exclusion type algorithm. After a two-step (internal/external validation, the best-performing classification models were used in a consensus predicting task for the identification of compounds named as “true” P-gp inhibitors, i.e., molecules able to inhibit P-gp without being effluxed by P-gp itself and simultaneously unable to activate the ATPase function.

Deinhibition of cardiac Na+-K+-ATPase after exposure to exogenous phospholipase A2

International Nuclear Information System (INIS)

Colvin, R.A.

1987-01-01

After 2 h of exogenous phospholipase A 2 (PLA 2 ) exposure, membrane phospholipid decreased from 3.22 +/- 0.31 to 1.06 +/- 0.13 μmol/mg (33% of control). All classes of phospholipid, except sphingomyelin, were hydrolyzed, whereas total cholesterol content was unaffected. Increases in nonesterified fatty acids (NEFA) were reflected primarily in oleic (18:1), linoleic (18:2), and arachidonic (20:4). Na + -K + -adenosinetriphosphatase (ATPase) activity was inhibited to 29% of control by 2 h of PLA 2 treatment, and this inhibition was reversed (albeit, not completely after 5 min of PLA 2 treatment) by removal of the hydrolysis products with 0.1% bovine serum albumin (BSA). In contrast, the apparent binding capacity for [ 3 H]ouabain was not affected by PLA 2 treatment. Unmasking of latent [ 3 H]ouabain binding by alamethicin was utilized to estimate changes in the proportion of sealed vesicles present before and after PLA 2 treatment. PLA 2 treatment resulted in a time-dependent loss of sealed vesicles that paralleled the time course of phospholipid hydrolysis and was not reversed by washing with BSA. These studies demonstrate that cardiac Na + -K + -ATPase activity is inhibited by accumulation of endogenously produced lysophospholipids and NEFA. In contrast, loss of vesicle integrity may result from both accumulation of endogenously produced hydrolysis products and membrane phospholipid depletion

K+-pNPPase located at the gastric epithelial cell exterior accounts for half of the total K+-pNPPase associated with Na+, K+-ATPase

International Nuclear Information System (INIS)

Nandi, J.; Levine, R.A.; Das, P.K.; Ray, T.K.

1986-01-01

Highly enriched and viable surface epithelial cells (SEC) isolated from rabbit stomach were used to study the ouabain-sensitive Na + , K + -ATPase and K + -pNPPase activities before and after lysis of the intact SEC. The viability of the SEC was monitored by erythrosin B dye exclusion. The function of the Na + , K + -ATPase pump was evaluated by measuring the ouabain-sensitive 86 Rb + uptake, which reached a steady-state (40-50 nmoles/10 6 cells) within 15-20 min at 37 0 C and maintained such a level for 60 min. Absence of any ouabain-insensitive H + , K + -ATPase activity in the lysed cells ensured negligible contamination from parietal cells. The intact SEC showed no Na + , K + -ATPase and insignificant Mg +2 -ATPase activity. However, a significant K + -pNPPase (0.93 +/- .034 μmoles/h.10 6 cells) responsive to oubain inhibition was demonstrated by localizing its activity to the cell surface exterior. The lysed SEC, on the other hand, demonstrated both the ouabain-sensitive Na + , K + -ATPase and K + -pNPPase activities and were 1.14 and 1.84 μmoles/h.10 6 cells, respectively. The data demonstrated the ATP hydrolytic site of the Na + K + ATPase to be facing the cytosol while the associated K + -pNPPase to be distributed equally across both sides of the plasma membrane. The data are consistent with a recent model of the monovalent cation transporting ATPase systems reported in gastric microsomes

Functional analysis of a potential regulatory K+-binding site in the Na+, K+-ATPase

DEFF Research Database (Denmark)

Schack, Vivien Rodacker; Vilsen, Bente

The Na+, K+-ATPase functions by actively transporting 3 Na+ ions out of and 2 K+ ions into the cell, thereby creating ion gradients crucial for many physiological processes. Recently, a combined structural and functional study of the closely related Ca2+-ATPase indicated the presence...... of a regulatory K+-binding site in the P-domain of the enzyme, identifying E732 as being of particular importance (Sorensen, Clausen et al. 2004). In addition, P709 is thought to play a significant role in the structural organization of this site. Both E732 and P709 are highly conserved among P-type ATPases (E732...... is present as either glutamic acid or aspartic acid), which supports their importance and additionally raises the question whether this site may play a general role among P-type ATPases. In Na+, K+-ATPase, K+ functions directly as a substrate for membrane binding sites, however, an additional regulatory...

Bicarbonate-regulated adenylyl cyclase (sAC) is a sensor that regulates pH-dependent V-ATPase recycling.

Science.gov (United States)

Pastor-Soler, Nuria; Beaulieu, Valerie; Litvin, Tatiana N; Da Silva, Nicolas; Chen, Yanqiu; Brown, Dennis; Buck, Jochen; Levin, Lonny R; Breton, Sylvie

2003-12-05

Modulation of environmental pH is critical for the function of many biological systems. However, the molecular identity of the pH sensor and its interaction with downstream effector proteins remain poorly understood. Using the male reproductive tract as a model system in which luminal acidification is critical for sperm maturation and storage, we now report a novel pathway for pH regulation linking the bicarbonate activated soluble adenylyl cyclase (sAC) to the vacuolar H+ATPase (V-ATPase). Clear cells of the epididymis and vas deferens contain abundant V-ATPase in their apical pole and are responsible for acidifying the lumen. Proton secretion is regulated via active recycling of V-ATPase. Here we demonstrate that this recycling is regulated by luminal pH and bicarbonate. sAC is highly expressed in clear cells, and apical membrane accumulation of V-ATPase is triggered by a sAC-dependent rise in cAMP in response to alkaline luminal pH. As sAC is expressed in other acid/base transporting epithelia, including kidney and choroid plexus, this cAMP-dependent signal transduction pathway may be a widespread mechanism that allows cells to sense and modulate extracellular pH.

Biochemical Characterization of P4-ATPase Mutations Associated with Intrahepatic Cholestatic Disease

DEFF Research Database (Denmark)

Gantzel, Rasmus; Vestergaard, Anna Lindeløv; Mikkelsen, Stine

The cholestatic disorders progressive familial intrahepatic cholestasis type 1 (PFIC1, also referred to as Byler’s disease) and benign recurrent intrahepatic cholestasis type 1 (BRIC1) are caused by mutation of the P4-ATPase ATP8B1. The substrate of ATP8B1 is very likely to be phosphatidylserine...... families have been investigated, and more than 50 distinct disease mutations have been identified, with roughly half being missense mutations. In this project we try to answer the question whether PFIC1 mutations are generally more disturbing than BRIC1 mutations with respect to expression, structural...... stability and function. We investigate the mutations in our well functioning system of ATP8A2, being expressed in mammalian HEK293T cells, affinity-purified, and reconstituted in lipid vesicles. Well-known mutations from both groups of patients have been selected for study. I91P in ATP8A2 (L127P in ATP8B1...

Biochemical characterization of P4-ATPase mutations associated with Intrahepatic Cholestatic Disease

DEFF Research Database (Denmark)

Gantzel, Rasmus; Vestergaard, Anna Lindeløv; Mikkelsen, Stine

Progressive familial intrahepatic cholestasis type 1 (PFIC1) and benign recurrent intrahepatic cholestasis type 1 (BRIC1) are caused by mutation of the P4-ATPase ATP8B1 that flips phospholipid from the exoplasmic leaflet to the cytoplasmic leaflet of canalicular membranes. It is hypothesized...... that PFIC1 mutations are the most disturbing with respect to expression, structural stability and/or function. Although recent data indicates that the specific phospholipid substrate of ATP8B1 is phosphatidylcholine (PC) [1] whereas ATP8A2 flips phosphatidylserine (PS) and phosphatidylethanolamine (PE......), there may be several mechanistic similarities between ATP8B1 and ATP8A2, and here we investigate known disease mutations using our well-functioning methodology for expression, affinity purification and assay of the partial reactions of ATP8A2. Mutations I91P (L127P in ATP8B1) and L308F (I344F) are located...

Vacuolar ATPases, like F1,F0-ATPases, show a strong dependence of the reaction velocity on the binding of more than one ATP per enzyme

International Nuclear Information System (INIS)

Kasho, V.N.; Boyer, P.D.

1989-01-01

Recent studies with vacuolar ATPases have shown that multiple copies catalytic subunits are present and that these have definite sequence homology with catalytic subunits of the F 1 , F 0 -ATPases. Experiments are reported that assess whether the vacuolar ATPases may have the unusual catalytic cooperativity with sequential catalytic site participation as in the binding change mechanism for the F 1 ,F 0 -ATPases. The extent of reversal of bound ATP hydrolysis to bound ADP and P i as medium ATP concentration was lowered was determined by 18 O-exchange measurements for yeast and neurospora vacuolar ATPases. The results show a pronounced increase in the extent of water oxygen incorporation into the P i formed as ATP concentration is decreased to the micromolar range. The F 1 ,F 0 -ATPase from neurospora mitochondria showed an event more pronounced modulation, similar to that of other F 1 -type ATPases. The vacuolar ATPases thus appear to have a catalytic mechanism quite analogous to that of the F 1 ,F 0 -ATPases

Inter-ring rotations of AAA ATPase p97 revealed by electron cryomicroscopy.

Science.gov (United States)

Yeung, Heidi O; Förster, Andreas; Bebeacua, Cecilia; Niwa, Hajime; Ewens, Caroline; McKeown, Ciarán; Zhang, Xiaodong; Freemont, Paul S

2014-03-05

The type II AAA+ protein p97 is involved in numerous cellular activities, including endoplasmic reticulum-associated degradation, transcription activation, membrane fusion and cell-cycle control. These activities are at least in part regulated by the ubiquitin system, in which p97 is thought to target ubiquitylated protein substrates within macromolecular complexes and assist in their extraction or disassembly. Although ATPase activity is essential for p97 function, little is known about how ATP binding or hydrolysis is coupled with p97 conformational changes and substrate remodelling. Here, we have used single-particle electron cryomicroscopy (cryo-EM) to study the effect of nucleotides on p97 conformation. We have identified conformational heterogeneity within the cryo-EM datasets from which we have resolved two major p97 conformations. A comparison of conformations reveals inter-ring rotations upon nucleotide binding and hydrolysis that may be linked to the remodelling of target protein complexes.

Mitotic phosphorylation of VCIP135 blocks p97ATPase-mediated Golgi membrane fusion

Energy Technology Data Exchange (ETDEWEB)

Totsukawa, Go; Matsuo, Ayaka; Kubota, Ayano; Taguchi, Yuya; Kondo, Hisao, E-mail: hk228@med.kyushu-u.ac.jp

2013-04-05

Highlights: •VCIP135 is mitotically phosphorylated on Threonine-760 and Serine-767 by Cdc2. •Phosphorylated VCIP135 does not bind to p97ATPase. •The phosphorylation of VCIP135 inhibits p97ATPase-mediated Golgi membrane fusion. -- Abstract: In mammals, the Golgi apparatus is disassembled early mitosis and reassembled at the end of mitosis. For Golgi disassembly, membrane fusion needs to be blocked. Golgi biogenesis requires two distinct p97ATPase-mediated membrane fusion, the p97/p47 and p97/p37 pathways. We previously reported that p47 phosphorylation on Serine-140 and p37 phosphorylation on Serine-56 and Threonine-59 result in mitotic inhibition of the p97/p47 and the p97/p37 pathways, respectively [11,14]. In this study, we show another mechanism of mitotic inhibition of p97-mediated Golgi membrane fusion. We clarified that VCIP135, an essential factor in both p97 membrane fusion pathways, is phosphorylated on Threonine-760 and Serine-767 by Cdc2 at mitosis and that this phosphorylated VCIP135 does not bind to p97. An in vitro Golgi reassembly assay revealed that VCIP135(T760E, S767E), which mimics mitotic phosphorylation, caused no cisternal regrowth. Our results indicate that the phosphorylation of VCIP135 on Threonine-760 and Serine-767 inhibits p97-mediated Golgi membrane fusion at mitosis.

Structure and function of the AAA+ ATPase p97/Cdc48p.

Science.gov (United States)

Xia, Di; Tang, Wai Kwan; Ye, Yihong

2016-05-25

p97 (also known as valosin-containing protein (VCP) in mammals or Cdc48p in Saccharomyces cerevisiae) is an evolutionarily conserved ATPase present in all eukaryotes and archaebacteria. In conjunction with a collection of cofactors and adaptors, p97/Cdc48p performs an array of biological functions mostly through modulating the stability of 'client' proteins. Using energy from ATP hydrolysis, p97/Cdc48p segregates these molecules from immobile cellular structures such as protein assemblies, membrane organelles, and chromatin. Consequently, the released polypeptides can be efficiently degraded by the ubiquitin proteasome system or recycled. This review summarizes our current understanding of the structure and function of this essential cellular chaperoning system. Published by Elsevier B.V.

Vacuolar-type H⁺-ATPase and Na⁺, K⁺-ATPase expression in gills of Atlantic salmon (Salmo salar) during isolated and combined exposure to hyperoxia and hypercapnia in fresh water

DEFF Research Database (Denmark)

Seidelin, Michel; Brauner, Colin J; Jensen, Frank Bo

2001-01-01

Changes in branchial vacuolar-type H+-ATPase B-subunit mRNA and Na+, K+-ATPase alpha- and beta-subunit mRNA and ATP hydrolytic activity were examined in smolting Atlantic salmon exposed to hyperoxic and/or hypercapnic fresh water. Pre-smolts, smolts, and post-smolts were exposed for 1 to 4 days...... Na+, K+-ATPase activity was generally unaffected by the experimental treatments. We suggest that the reduced expression of branchial vacuolar-type H(+)-ATPase B-subunit mRNA observed during internal hypercapnic acidosis may lead to reduction of functional V-type H+-ATPase abundance as a compensatory...

Asn792 participates in the hydrogen bond network around the K+-binding pocket of gastric H,K-ATPase.

NARCIS (Netherlands)

Swarts, H.G.P.; Koenderink, J.B.; Willems, P.H.G.M.; Krieger, E.; Pont, J.J.H.H.M. de

2005-01-01

Asn792 present in M5 of gastric H,K-ATPase is highly conserved within the P-type ATPase family. A direct role in K+ binding was postulated for Na,K-ATPase but was not found in a recent model for gastric H,K-ATPase (Koenderink, J. B., Swarts, H. G. P., Willems, P. H. G. M., Krieger, E., and De Pont,

Specific inhibition of p97/VCP ATPase and kinetic analysis demonstrate interaction between D1 and D2 ATPase domains.

Science.gov (United States)

Chou, Tsui-Fen; Bulfer, Stacie L; Weihl, Conrad C; Li, Kelin; Lis, Lev G; Walters, Michael A; Schoenen, Frank J; Lin, Henry J; Deshaies, Raymond J; Arkin, Michelle R

2014-07-29

The p97 AAA (ATPase associated with diverse cellular activities), also called VCP (valosin-containing protein), is an important therapeutic target for cancer and neurodegenerative diseases. p97 forms a hexamer composed of two AAA domains (D1 and D2) that form two stacked rings and an N-terminal domain that binds numerous cofactor proteins. The interplay between the three domains in p97 is complex, and a deeper biochemical understanding is needed in order to design selective p97 inhibitors as therapeutic agents. It is clear that the D2 ATPase domain hydrolyzes ATP in vitro, but whether D1 contributes to ATPase activity is controversial. Here, we use Walker A and B mutants to demonstrate that D1 is capable of hydrolyzing ATP and show for the first time that nucleotide binding in the D2 domain increases the catalytic efficiency (kcat/Km) of D1 ATP hydrolysis 280-fold, by increasing kcat 7-fold and decreasing Km about 40-fold. We further show that an ND1 construct lacking D2 but including the linker between D1 and D2 is catalytically active, resolving a conflict in the literature. Applying enzymatic observations to small-molecule inhibitors, we show that four p97 inhibitors (DBeQ, ML240, ML241, and NMS-873) have differential responses to Walker A and B mutations, to disease-causing IBMPFD mutations, and to the presence of the N domain binding cofactor protein p47. These differential effects provide the first evidence that p97 cofactors and disease mutations can alter p97 inhibitor potency and suggest the possibility of developing context-dependent inhibitors of p97. Copyright © 2014 Elsevier Ltd. All rights reserved.

Double-stranded DNA-dependent ATPase Irc3p is directly involved in mitochondrial genome maintenance.

Science.gov (United States)

Sedman, Tiina; Gaidutšik, Ilja; Villemson, Karin; Hou, YingJian; Sedman, Juhan

2014-12-01

Nucleic acid-dependent ATPases are involved in nearly all aspects of DNA and RNA metabolism. Previous studies have described a number of mitochondrial helicases. However, double-stranded DNA-dependent ATPases, including translocases or enzymes remodeling DNA-protein complexes, have not been identified in mitochondria of the yeast Saccharomyces cerevisae. Here, we demonstrate that Irc3p is a mitochondrial double-stranded DNA-dependent ATPase of the Superfamily II. In contrast to the other mitochondrial Superfamily II enzymes Mss116p, Suv3p and Mrh4p, which are RNA helicases, Irc3p has a direct role in mitochondrial DNA (mtDNA) maintenance. Specific Irc3p-dependent mtDNA metabolic intermediates can be detected, including high levels of double-stranded DNA breaks that accumulate in irc3Δ mutants. irc3Δ-related topology changes in rho- mtDNA can be reversed by the deletion of mitochondrial RNA polymerase RPO41, suggesting that Irc3p counterbalances adverse effects of transcription on mitochondrial genome stability. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

The parietal cell gastric H, K-ATPase also functions as the Na, K-ATPase and Ca-ATPase in altered states.

Science.gov (United States)

Ray, Tushar

2013-01-01

This article offers an explanation for the apparent lack of Na, K-ATPase activity in parietal cells although ouabain has been known to inhibit gastric acid secretion since 1962. The gastric H, K-ATPase (proton-pump) seems to be acting in altered states, thus behaving like a Na, K-ATPase (Na-pump) and/or Ca-ATPase (Ca-pump) depending on cellular needs.  This conclusion is based on the following findings. First, parietal cell fractions do not exhibit Na, K-ATPase activity at pH 7.0 but do at pH 8.5. Second, the apical plasma membrane (APM) fraction exhibits a (Ca or Mg)-ATPase activity with negligible H, K-ATPase activity. However, when assayed with Mg alone in presence of the 80 k Da cytosolic proton-pump activator (HAF), the APM fraction reveals remarkably high H, K-ATPase activity, suggesting the observed low affinity of Ca (or Mg)-ATPase is an altered state of the latter. Third, calcium (between 1 and 4 µM) shows both stimulation and inhibition of the HAF-stimulated H, K-ATPase depending on its concentration, revealing a close interaction between the  proton-pump activator and local Ca concentration in gastric H, K-ATPase function. Such interactions suggest that Ca is acting as a terminal member of the intracellular signaling system for the HAF-regulated proton-pump. It appears that during resting state, the HAF-associated H, K-ATPase remains inhibited by Ca (>1 µM) and, prior to resumption of acid secretion the gastric H, K-ATPase acts temporarily as a Ca-pump for removing excess Ca from its immediate environment. This conclusion is consistent with the recent reports of immunochemical co-localization of the gastric H, K-ATPase and Ca-ATPase by superimposition in parietal cells, and a transitory efflux of Ca immediately preceding the onset of acid secretion. These new perspectives on proton-pump function would open new avenues for a fuller understanding of the intracellular regulation of the ubiquitous Na-pump.

Epinephrine modulates Na+/K+ ATPase activity in Caco-2 cells via Src, p38MAPK, ERK and PGE2.

Directory of Open Access Journals (Sweden)

Layla El Moussawi

Full Text Available Epinephrine, a key stress hormone, is known to affect ion transport in the colon. Stress has been associated with alterations in colonic functions leading to changes in water movements manifested as diarrhea or constipation. Colonic water movement is driven by the Na+-gradient created by the Na+/K+-ATPase. Whether epinephrine acts via an effect on the Na+/K+-ATPase hasn't been studied before. The aim of this work was to investigate the effect of epinephrine on the Na+/K+-ATPase and to elucidate the signaling pathway involved using CaCo-2 cells as a model. The activity of the Na+/K+-ATPase was assayed by measuring the amount of inorganic phosphate released in presence and absence of ouabain, a specific inhibitor of the enzyme. Epinephrine, added for 20 minutes, decreased the activity of the Na+/K+-ATPase by around 50%. This effect was found to be mediated by α2 adrenergic receptors as it was fully abolished in the presence of yohimbine an α2-blocker, but persisted in presence of other adrenergic antagonists. Furthermore, treatment with Rp-cAMP, a PKA inhibitor, mimicked epinephrine's negative effect and didn't result in any additional inhibition when both were added simultaneously. Treatment with indomethacin, PP2, SB202190, and PD98059, respective inhibitors of COX enzymes, Src, p38MAPK, and ERK completely abrogated the effect of epinephrine. The effect of epinephrine did not appear also in presence of inhibitors of all four different types of PGE2 receptors. Western blot analysis revealed an epinephrine-induced increase in the phosphorylation of p38 MAPK and ERK that disappeared in presence of respectively PP2 and SB2020190. In addition, an inhibitory effect, similar to that of epinephrine's, was observed upon incubation with PGE2. It was concluded that epinephrine inhibits the Na+/K+-ATPase by the sequential activation of α2 adrenergic receptors, Src, p38MAPK, and ERK leading to PGE2 release.

Phosphosite mapping of P-type plasma membrane H+-ATPase in homologous and heterologous environments

DEFF Research Database (Denmark)

Rudashevskaya, Elena; Ye, Juanying; Jensen, Ole Nørregaard

2012-01-01

Phosphorylation is an important posttranslational modification of proteins in living cells and primarily serves regulatory purposes. Several methods were employed for isolating phosphopeptides from proteolytically digested plasma membranes of Arabidopsis thaliana. After a mass spectrometric...... of the phosphosites identified in AHA2 were identical in the plant and fungal systems even though none of the target sequences in AHA2 show homology to proteins of the fungal host. These findings suggest an unexpected accessibility of the terminal regulatory domain of plasma membrane H(+)-ATPase to protein kinase...... analysis of the resulting peptides we could identify 10 different phosphorylation sites in plasma membrane H(+)-ATPases AHA1, AHA2, AHA3, and AHA4/11, five of which have not been reported before, bringing the total number of phosphosites up to 11, which is substantially higher than reported so far for any...

Mutant p97 exhibits species-specific changes of its ATPase activity and compromises the UBXD9-mediated monomerisation of p97 hexamers.

Science.gov (United States)

Rijal, Ramesh; Arhzaouy, Khalid; Strucksberg, Karl-Heinz; Cross, Megan; Hofmann, Andreas; Schröder, Rolf; Clemen, Christoph S; Eichinger, Ludwig

2016-01-01

p97 (VCP) is a homo-hexameric triple-A ATPase that exerts a plethora of cellular processes. Heterozygous missense mutations of p97 cause at least five human neurodegenerative disorders. However, the specific molecular consequences of p97 mutations are hitherto widely unknown. Our in silico structural models of human and Dictyostelium p97 showed that the disease-causing human R93C, R155H, and R155C as well as Dictyostelium R154C, E219K, R154C/E219K p97 mutations constitute variations in surface-exposed locations. In-gel ATPase activity measurements of p97 monomers and hexamers revealed significant mutation- and species-specific differences. While all human p97 mutations led to an increase in ATPase activity, no changes could be detected for the Dictyostelium R154C mutant, which is orthologous to human R155C. The E219K mutation led to an almost complete loss of activity, which was partially recuperated in the R154C/E219K double-mutant indicating p97 inter-domain communication. By means of co-immunoprecipitation experiments we identified an UBX-domain containing Dictyostelium protein as a novel p97 interaction partner. We categorized all UBX-domain containing Dictyostelium proteins and named the interaction partner UBXD9. Pull-down assays and surface plasmon resonance analyses of Dictyostelium UBXD9 or the human orthologue TUG/ASPL/UBXD9 demonstrated direct interactions with p97 as well as species-, mutation- and ATP-dependent differences in the binding affinities. Sucrose density gradient assays revealed that both human and Dictyostelium UBXD9 proteins very efficiently disassembled wild-type, but to a lesser extent mutant p97 hexamers into monomers. Our results are consistent with a scenario in which p97 point mutations lead to differences in enzymatic activities and molecular interactions, which in the long-term result in a late-onset and progressive multisystem disease. Copyright © 2016 The Authors. Published by Elsevier GmbH.. All rights reserved.

Relationship between serum leptin levels, ATPase activity of erythrocyte membrance and development of diabetic nephropathy in patients with DM2

International Nuclear Information System (INIS)

Wang Yuming

2009-01-01

Objective: To study the possible mechanism of development of nephrosis affected by changes of serum leptin levels and alteration of activities of Na + K + -ATPase and Ca 2+ Mg 2+ -ATPase of erythrocyte membrane in patients with type 2 diabetes(DM2). Methods: Serum leptin levels (with RIA) and erythrocyte membrane Na + K + -ATPase and Ca 2+ Mg 2+ -ATPase activitities (with Reinila method) were determined in 40 DM2 patients without nephropathy, 32 DM2 patients with nephropathy and 35 controls. Results Serum leptin levels were significantly higher in the diabetics as a whole than those in controls (P + K + -ATPase and Ca 2+ Mg 2+ -ATPase activities were significantly lower (P<0.01). Among the diabetic patients, the serum leptin levels in patients without nephrosis (P<0.05), but the RBC membrance ATPase activities were significantly lower(P<0.05). Conclusion: Development of type 2 diabetes nephrosis might be correlated with the high serum leptin level and decreased ATPase activities of erythrocite membrane. (authors)

Low pH-induced changes of antioxidant enzyme and ATPase activities in the roots of rice (Oryza sativa L. seedlings.

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Yi-Kai Zhang

Full Text Available Soil acidification is the main problem in the current rice production. Here, the effects of low pH on the root growth, reactive oxygen species metabolism, plasma membrane functions, and the transcript levels of the related genes were investigated in rice seedlings (Oryza sativa L. in a hydroponic system at pH 3.5, 4.5, and 5.5. There were two hybrid rice cultivars in this trial, including Yongyou 12 (YY12, a japonica hybrid and Zhongzheyou 1 (ZZY1, an indica hybrid. Higher H+ activity markedly decreased root length, the proportion of fine roots, and dry matter production, but induced a significant accumulation of hydrogen peroxide (H2O2, and led to serious lipid peroxidation in the roots of the two varieties. The transcript levels of copper/zinc superoxide dismutase 1 (Cu/Zn SOD1, copper/zinc superoxide dismutase 2 (Cu/Zn SOD2, catalase A (CATA and catalase B (CATB genes in YY12 and ZZY1 roots were significantly down-regulated after low pH exposure for two weeks. Meanwhile, a significant decrease was observed in the expression of the P-type Ca2+-ATPases in roots at pH 3.5. The activities of antioxidant enzymes (SOD, CAT and plasma membrane (PM Ca2+-ATPase in the two varieties were dramatically inhibited by strong rhizosphere acidification. However, the expression levels of ascorbate peroxidase 1 (APX1 and PM H+-ATPase isoform 7 were up-regulated under H+ stress compared with the control. Significantly higher activities of APX and PM H+-ATPase could contribute to the adaptation of rice roots to low pH.

Porphyromonas gingivalis is highly sensitive to inhibitors of a proton-pumping ATPase.

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Sekiya, Mizuki; Shimoyama, Yu; Ishikawa, Taichi; Sasaki, Minoru; Futai, Masamitsu; Nakanishi-Matsui, Mayumi

2018-04-15

Porphyromonas gingivalis is a well-known Gram-negative bacterium that causes periodontal disease. The bacterium metabolizes amino acids and peptides to obtain energy. An ion gradient across its plasma membrane is thought to be essential for nutrient import. However, it is unclear whether an ion-pumping ATPase responsible for the gradient is required for bacterial growth. Here, we report the inhibitory effect of protonophores and inhibitors of a proton-pumping ATPase on the growth of P. gingivalis. Among the compounds examined, curcumin and citreoviridin appreciably reduced the bacterial growth. Furthermore, these compounds inhibited the ATPase activity in the bacterial membrane, where the A-type proton-pumping ATPase (A-ATPase) is located. This study suggests that curcumin and citreoviridin inhibit the bacterial growth by inhibiting the A-ATPase in the P. gingivalis membrane. Copyright © 2018 Elsevier Inc. All rights reserved.

Cardiac Glycoside Glucoevatromonoside Induces Cancer Type-Specific Cell Death

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Naira F. Z. Schneider

2018-03-01

Full Text Available Cardiac glycosides (CGs are natural compounds used traditionally to treat congestive heart diseases. Recent investigations repositioned CGs as potential anticancer agents. To discover novel cytotoxic CG scaffolds, we selected the cardenolide glucoevatromonoside (GEV out of 46 CGs for its low nanomolar anti-lung cancer activity. GEV presented reduced toxicity toward non-cancerous cell types (lung MRC-5 and PBMC and high-affinity binding to the Na+/K+-ATPase α subunit, assessed by computational docking. GEV-induced cell death was caspase-independent, as investigated by a multiparametric approach, and culminates in severe morphological alterations in A549 cells, monitored by transmission electron microscopy, live cell imaging and flow cytometry. This non-canonical cell death was not preceded or accompanied by exacerbation of autophagy. In the presence of GEV, markers of autophagic flux (e.g. LC3I-II conversion were impacted, even in presence of bafilomycin A1. Cell death induction remained unaffected by calpain, cathepsin, parthanatos, or necroptosis inhibitors. Interestingly, GEV triggered caspase-dependent apoptosis in U937 acute myeloid leukemia cells, witnessing cancer-type specific cell death induction. Differential cell cycle modulation by this CG led to a G2/M arrest, cyclin B1 and p53 downregulation in A549, but not in U937 cells. We further extended the anti-cancer potential of GEV to 3D cell culture using clonogenic and spheroid formation assays and validated our findings in vivo by zebrafish xenografts. Altogether, GEV shows an interesting anticancer profile with the ability to exert cytotoxic effects via induction of different cell death modalities.

31P NMR Spectroscopy Revealed Adenylate kinase-like Activity and Phosphotransferase-like Activity from F1-ATPase of Escherichia coli

International Nuclear Information System (INIS)

Kim, Hyun Won

2011-01-01

Adenylate kinase-like activity and phosphotransferase-like activity from F 1 -ATPase of Escherichia coli was revealed by 31 P NMR spectroscopy. Incubation of F 1 -ATPase with ADP in the presence of Mg 2+ shows the appearance of 31 P resonances from AMP and Pi, suggesting generation of AMP and ATP by adenylate kinase-like activity and the subsequent hydrolysis to Pi. Incubation of F1-ATPase with ADP in the presence of methanol shows additional peak from methyl phosphate, suggesting phosphotransferase-like activity of F 1 -ATPase. Both adenylate kinase-like activity and phosphotransferase-like activity has not been reported from F 1 -ATPase of Escherichia coli. 31 P NMR could be a valuable tool for the investigation of phosphorous related enzyme

The F1 -ATPase from Trypanosoma brucei is elaborated by three copies of an additional p18-subunit.

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Gahura, Ondřej; Šubrtová, Karolína; Váchová, Hana; Panicucci, Brian; Fearnley, Ian M; Harbour, Michael E; Walker, John E; Zíková, Alena

2018-02-01

The F-ATPases (also called the F 1 F o -ATPases or ATP synthases) are multi-subunit membrane-bound molecular machines that produce ATP in bacteria and in eukaryotic mitochondria and chloroplasts. The structures and enzymic mechanisms of their F 1 -catalytic domains are highly conserved in all species investigated hitherto. However, there is evidence that the F-ATPases from the group of protozoa known as Euglenozoa have novel features. Therefore, we have isolated pure and active F 1 -ATPase from the euglenozoan parasite, Trypanosoma brucei, and characterized it. All of the usual eukaryotic subunits (α, β, γ, δ, and ε) were present in the enzyme, and, in addition, two unique features were detected. First, each of the three α-subunits in the F 1 -domain has been cleaved by proteolysis in vivo at two sites eight residues apart, producing two assembled fragments. Second, the T. brucei F 1 -ATPase has an additional subunit, called p18, present in three copies per complex. Suppression of expression of p18 affected in vitro growth of both the insect and infectious mammalian forms of T. brucei. It also reduced the levels of monomeric and multimeric F-ATPase complexes and diminished the in vivo hydrolytic activity of the enzyme significantly. These observations imply that p18 plays a role in the assembly of the F 1 domain. These unique features of the F 1 -ATPase extend the list of special characteristics of the F-ATPase from T. brucei, and also, demonstrate that the architecture of the F 1 -ATPase complex is not strictly conserved in eukaryotes. © 2017 Federation of European Biochemical Societies.

The V-ATPase membrane domain is a sensor of granular pH that controls the exocytotic machinery.

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Poëa-Guyon, Sandrine; Ammar, Mohamed Raafet; Erard, Marie; Amar, Muriel; Moreau, Alexandre W; Fossier, Philippe; Gleize, Vincent; Vitale, Nicolas; Morel, Nicolas

2013-10-28

Several studies have suggested that the V0 domain of the vacuolar-type H(+)-adenosine triphosphatase (V-ATPase) is directly implicated in secretory vesicle exocytosis through a role in membrane fusion. We report in this paper that there was a rapid decrease in neurotransmitter release after acute photoinactivation of the V0 a1-I subunit in neuronal pairs. Likewise, inactivation of the V0 a1-I subunit in chromaffin cells resulted in a decreased frequency and prolonged kinetics of amperometric spikes induced by depolarization, with shortening of the fusion pore open time. Dissipation of the granular pH gradient was associated with an inhibition of exocytosis and correlated with the V1-V0 association status in secretory granules. We thus conclude that V0 serves as a sensor of intragranular pH that controls exocytosis and synaptic transmission via the reversible dissociation of V1 at acidic pH. Hence, the V-ATPase membrane domain would allow the exocytotic machinery to discriminate fully loaded and acidified vesicles from vesicles undergoing neurotransmitter reloading.

Modulation of the transducer function of Na+,K+-ATPase: new mechanism of heart remodeling.

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Lopatina, Ekaterina V; Kipenko, Anna V; Pasatetskaya, Natalia A; Penniyaynen, Valentina A; Krylov, Boris V

2016-10-01

Endogenous digitalis-like factors were found in the mammalian and human blood. It was the starting point for the elucidation of the new non-pumping function of the Na + ,K + -ATPase. It was previously well known that Na + ,K + -ATPase is a pharmacological target receptor for cardiac glycosides (J.C. Skou. 1957. Biochim. Biophys. Acta, 23: 394-401). We have investigated the trophotropic effects of such agents as ouabain, epinephrine, norepinephrine, atenolol, and comenic acid using the organotypic tissue culture combined with the reconstruction of optical cross sections and confocal microscopy. It was shown that the growth zone of organotypic culture forms a multidimensional structure. Our results indicate that the cardiac glycoside ouabain applied in endogenous concentrations (10 -8 , 10 -10 mol/L) can modulate transducer function of Na + ,K + -ATPase and control the cell growth and proliferation. It was also shown that Src-kinase is involved in "endogenous" ouabain activated intracellular pathways as a series unit. Epinephrine (10 -9 -10 -14 mol/L) and comenic acid (10 -6 -10 -10 mol/L) were demonstrated to modulate the growth of 10- to 12-day-old chicken embryo cardiac tissue explants in a dose-dependent manner. Epinephrine and comenic acid regulate growth and proliferation of the cardiac tissue via receptor-mediated modulation Na + ,K + -ATPase as a signal transducer. The trophotropic effects of the investigated agents specifically control the heart remodeling phenomenon.

Left ventricular wall stress and sarcoplasmic reticulum Ca(2+)-ATPase gene expression in renal hypertensive rats: dose-dependent effects of ACE inhibition and AT1-receptor blockade.

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Zierhut, W; Studer, R; Laurent, D; Kästner, S; Allegrini, P; Whitebread, S; Cumin, F; Baum, H P; de Gasparo, M; Drexler, H

1996-05-01

Cardiac hypertrophy is associated with altered Ca2+ handling and may predispose to the development of LV dysfunction and cardiac failure. At the cellular level, the re-expression of ANF represents a well-established marker of myocyte hypertrophy while the decreased expression of the sarcoplasmatic reticulum (SR) Ca(2+)-ATPase is thought o play a crucial role in the alterations of Ca2+ handling and LV function. We assessed the dose-dependent effect of chronic ACE inhibition or AT1 receptor blockade on cardiac function in relation to the cardiac expression of the SR Ca(2+)-ATPase and ANF. Renal hypertensive rats (2K-1C) were treated for 12 weeks with three different doses of the ACE inhibitor benazepril, the AT1-receptor antagonist valsartan (each drug 0.3, 3, and 10 mg/kg per day i.p.) or placebo. LV dimensions, hypertrophy and wall stress were determined in vivo by magnetic resonance imaging and the gene expressions of ANF and SR Ca(2+)-ATPase were quantified by Northern blot. Low doses of both drugs did not affect blood pressure, hypertrophy, systolic wall stress and the ANF and SR Ca(2+)-ATPase gene expression. High doses of each drug reduced systolic blood pressure, wall stress, and LV hypertrophy to a similar extent and to values comparable to normotensive, age-matched rats. In addition, high dose treatment reduced LV end-systolic and end-diastolic volume as compared to untreated 2K-1C animals and normalized the mRNA levels of both ANF and SR Ca(2+)-ATPase (as compared to normotensive animals). We conclude that in this model, high doses of ACE inhibition and AT1-receptor blockade are necessary to normalize systolic blood pressure, LV hypertrophy and systolic LV wall stress which, in turn, is associated with restoration of a normal cardiac phenotype with respect to SR Ca(2+)-ATPase and ANF and normalization of cardiac function.

ECA3, a Golgi-localized P_2A-type-ATPase, plays a crucial role in manganese nutrition in Arabidopsis

DEFF Research Database (Denmark)

Mills, Rebecca F.; Doherty, Melissa Louise; Lopez Marques, Rosa Laura

2008-01-01

and development, and transport processes play a key role in regulating their cellular levels. Arabidopsis (Arabidopsis thaliana) contains four P(2A)-type ATPase genes, AtECA1 to AtECA4, which are expressed in all major organs of Arabidopsis. To elucidate the physiological role of AtECA2 and AtECA3 in Arabidopsis...... not so striking because in this case all plants were severely affected. ECA3 partially restored the growth defect on high Mn of the yeast (Saccharomyces cerevisiae) pmr1 mutant, which is defective in a Golgi Ca/Mn pump (PMR1), and the yeast K616 mutant (Deltapmc1 Deltapmr1 Deltacnb1), defective in Golgi...

Cardiac Calcium ATPase Dimerization Measured by Cross-Linking and Fluorescence Energy Transfer.

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Blackwell, Daniel J; Zak, Taylor J; Robia, Seth L

2016-09-20

The cardiac sarco/endoplasmic reticulum calcium ATPase (SERCA) establishes the intracellular calcium gradient across the sarcoplasmic reticulum membrane. It has been proposed that SERCA forms homooligomers that increase the catalytic rate of calcium transport. We investigated SERCA dimerization in rabbit left ventricular myocytes using a photoactivatable cross-linker. Western blotting of cross-linked SERCA revealed higher-molecular-weight species consistent with SERCA oligomerization. Fluorescence resonance energy transfer measurements in cells transiently transfected with fluorescently labeled SERCA2a revealed that SERCA readily forms homodimers. These dimers formed in the absence or presence of the SERCA regulatory partner, phospholamban (PLB) and were unaltered by PLB phosphorylation or changes in calcium or ATP. Fluorescence lifetime data are compatible with a model in which PLB interacts with a SERCA homodimer in a stoichiometry of 1:2. Together, these results suggest that SERCA forms constitutive homodimers in live cells and that dimer formation is not modulated by SERCA conformational poise, PLB binding, or PLB phosphorylation. Copyright © 2016 Biophysical Society. Published by Elsevier Inc. All rights reserved.

Expression of Na,K-ATPase and H,K-ATPase Isoforms with the Baculovirus Expression System

NARCIS (Netherlands)

Koenderink, J.B.; Swarts, H.G.

2016-01-01

P-type ATPases can be expressed in several cell systems. The baculovirus expressions system uses an insect virus to enter and express proteins in Sf9 insect cells. This expression system is a lytic system in which the cells will die a few days after viral infection. Subsequently, the expressed

Genome-wide analysis of wheat calcium ATPases and potential role of selected ACAs and ECAs in calcium stress.

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Aslam, Roohi; Williams, Lorraine E; Bhatti, Muhammad Faraz; Virk, Nasar

2017-10-27

P 2 - type calcium ATPases (ACAs-auto inhibited calcium ATPases and ECAs-endoplasmic reticulum calcium ATPases) belong to the P- type ATPase family of active membrane transporters and are significantly involved in maintaining accurate levels of Ca 2+ , Mn 2+ and Zn 2+ in the cytosol as well as playing a very important role in stress signaling, stomatal opening and closing and pollen tube growth. Here we report the identification and possible role of some of these ATPases from wheat. In this study, ACA and ECA sequences of six species (belonging to Poaceae) were retrieved from different databases and a phylogenetic tree was constructed. A high degree of evolutionary relatedness was observed among P 2 sequences characterized in this study. Members of the respective groups from different plant species were observed to fall under the same clade. This pattern highlights the common ancestry of P 2- type calcium ATPases. Furthermore, qRT-PCR was used to analyse the expression of selected ACAs and ECAs from Triticum aestivum (wheat) under calcium toxicity and calcium deficiency. The data indicated that expression of ECAs is enhanced under calcium stress, suggesting possible roles of these ATPases in calcium homeostasis in wheat. Similarly, the expression of ACAs was significantly different in plants grown under calcium stress as compared to plants grown under control conditions. This gives clues to the role of ACAs in signal transduction during calcium stress in wheat. Here we concluded that wheat genome consists of nine P 2B and three P 2A -type calcium ATPases. Moreover, gene loss events in wheat ancestors lead to the loss of a particular homoeolog of a gene in wheat. To elaborate the role of these wheat ATPases, qRT-PCR was performed. The results indicated that when plants are exposed to calcium stress, both P 2A and P 2B gene expression get enhanced. This further gives clues about the possible role of these ATPases in wheat in calcium management. These findings can be

AtHMA3, a P1B-ATPase allowing Cd/Zn/Co/Pb vacuolar storage in Arabidopsis.

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Morel, Mélanie; Crouzet, Jérôme; Gravot, Antoine; Auroy, Pascaline; Leonhardt, Nathalie; Vavasseur, Alain; Richaud, Pierre

2009-02-01

The Arabidopsis (Arabidopsis thaliana) Heavy Metal Associated3 (AtHMA3) protein belongs to the P1B-2 subgroup of the P-type ATPase family, which is involved in heavy metal transport. In a previous study, we have shown, using heterologous expression in the yeast Saccharomyces cerevisiae, that in the presence of toxic metals, AtHMA3 was able to phenotypically complement the cadmium/lead (Cd/Pb)-hypersensitive strain ycf1 but not the zinc (Zn)-hypersensitive strain zrc1. In this study, we demonstrate that AtHMA3 in planta is located in the vacuolar membrane, with a high expression level in guard cells, hydathodes, vascular tissues, and the root apex. Confocal imaging in the presence of the Zn/Cd fluorescent probe BTC-5N revealed that AtHMA3 participates in the vacuolar storage of Cd. A T-DNA insertional mutant was found more sensitive to Zn and Cd. Conversely, ectopic overexpression of AtHMA3 improved plant tolerance to Cd, cobalt, Pb, and Zn; Cd accumulation increased by about 2- to 3-fold in plants overexpressing AtHMA3 compared with wild-type plants. Thus, AtHMA3 likely plays a role in the detoxification of biological (Zn) and nonbiological (Cd, cobalt, and Pb) heavy metals by participating in their vacuolar sequestration, an original function for a P1B-2 ATPase in a multicellular eukaryote.

Ouabain affects cell migration via Na,K-ATPase-p130cas and via nucleus-centrosome association.

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Young Ou

Full Text Available Na,K-ATPase is a membrane protein that catalyzes ATP to maintain transmembrane sodium and potassium gradients. In addition, Na,K-ATPase also acts as a signal-transducing receptor for cardiotonic steroids such as ouabain and activates a number of signalling pathways. Several studies report that ouabain affects cell migration. Here we used ouabain at concentrations far below those required to block Na,K-ATPase pump activity and show that it significantly reduced RPE cell migration through two mechanisms. It causes dephosphorylation of a 130 kD protein, which we identify as p130cas. Src is involved, because Src inhibitors, but not inhibitors of other kinases tested, caused a similar reduction in p130cas phosphorylation and ouabain increased the association of Na,K-ATPase and Src. Knockdown of p130cas by siRNA reduced cell migration. Unexpectedly, ouabain induced separation of nucleus and centrosome, also leading to a block in cell migration. Inhibitor and siRNA experiments show that this effect is mediated by ERK1,2. This is the first report showing that ouabain can regulate cell migration by affecting nucleus-centrosome association.

Activation of proteolytic enzymes and depression of the sarcolemmal Na+/K+-ATPase in ischemia-reperfused heart may be mediated through oxidative stress.

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Singh, Raja B; Hryshko, Larry; Freed, Darren; Dhalla, Naranjan S

2012-02-01

We tested whether the activation of proteolytic enzymes, calpain, and matrix metalloproteinases (MMPs) during ischemia-reperfusion (I/R) is mediated through oxidative stress. For this purpose, isolated rat hearts were subjected to a 30 min global ischemia followed by a 30 min reperfusion. Cardiac function was monitored and the activities of Na(+)/K(+)-ATPase, Mg(2+)-ATPase, calpain, and MMP were measured. Depression of cardiac function and Na(+)/K(+)-ATPase activity in I/R hearts was associated with increased calpain and MMP activities. These alterations owing to I/R were similar to those observed in hearts perfused with hypoxic medium, H(2)O(2) and xanthine plus xanthine oxidase. The I/R-induced changes were attenuated by ischemic preconditioning as well as by perfusing the hearts with N-acetylcysteine or mercaptopropionylglycine. Inhibition of MMP activity in hearts treated with doxycycline depressed the I/R-induced changes in cardiac function and Na(+)/K(+)-ATPase activity without affecting the calpain activation. On the other hand, inhibition of calpain activity upon treatment with leupeptin or MDL 28170 significantly reduced the MMP activity in addition to attenuating the I/R-induced alterations in cardiac function and Na(+)/K(+)-ATPase activity. These results suggest that the I/R-induced depression in Na(+)/K(+)-ATPase and cardiac function may be a consequence of the increased activities of both calpain and MMP because of oxidative stress in the heart.

Structural and functional studies of heavy metal ATPases

DEFF Research Database (Denmark)

Sitsel, Oleg

2015-01-01

to handle heavy metal ions. LpCopA is then compared to its two human homologues ATP7A and ATP7B, which cause the severe Menkes and Wilson diseases when malfunctioning. The differences between the three proteins are described and disease-causing mutations in the human proteins are analyzed. The crystal......Copper and zinc are trace elements that are crucial for the well-being of all cells and are an indispensable part of many proteins. At the same time, the intracellular levels of these metals require careful regulation, as an excess or deficiency may be lethal. P1B-ATPases are key players in Cu......+ and Zn2+ homeostasis that belong to the superfamily of P-type ATPases, transmembrane proteins which are present in virtually all lifeforms, with functions ranging from membrane potential generation to muscle relaxation. The goal of this thesis is to improve our understanding of P1B-ATPases by focusing...

Isolation and characterization of DNA-dependent ATPases from the Novikoff Hepatoma

International Nuclear Information System (INIS)

Thomas, D.C.

1984-01-01

Four DNA-dependent ATPases have been purified to apparent homogeneity from extracts of the Novikoff Hepatoma, and named ATPases II, III, IV, and V. The physical and enzymological properties of ATPases II, III, and V are nearly identical, and from tryptic peptide mapping these proteins were determined to be related, though they are still chromatographically distinct; all appear to be dimers. ATPaseIV is unique among the ATPases, and is probably a monomer. ATPase V appears much more stable to thermal inactivation than the similar curves generated by ATPases II, and III. ATPase IV, however, projects of a heat-inactivation curve intermediate to these two types. ATPase II is labelled to a much higher degree than the others when treated with a heterologous protein kinase using gamma-[ 32 P]-ATP. When ATPase II was treated with this kinase, and subsequently run over a DNA-cellulose column, the profile of ATPase II was found to contain small peaks of activity in the positions where ATPases III and V normally elute, suggesting that ATPase II may be a dephosphorylated form of the other two. The ATPases have been extensively characterized with respect to reaction products and requirements, substrate utilization, DNA effector requirements, and effects of ATP analogs

Thermophilic P-loop transport ATPases : Enzyme function and energetics at high temperature

NARCIS (Netherlands)

Pretz, Monika Gyöngyi

2007-01-01

Primary transport ATPases are divided into several superfamilies; amongst others including ATPases of the ABC transporter superfamily, the F-ATPase superfamily or the motor ATPases of the General Secretory (Sec) pathway. Motor proteins from these superfamilies show a low sequence similarity, except

Protein kinase C-mediated ATP stimulation of Na(+)-ATPase activity in LLC-PK1 cells involves a P2Y2 and/or P2Y4 receptor.

Science.gov (United States)

Wengert, M; Ribeiro, M C; Abreu, T P; Coutinho-Silva, R; Leão-Ferreira, L R; Pinheiro, A A S; Caruso-Neves, C

2013-07-15

ATP-activated P2Y receptors play an important role in renal sodium excretion. The aim of this study was to evaluate the modulation of ATPase-driven sodium reabsorption in the proximal tubule by ATP or adenosine (Ado). LLC-PK1 cells, a model of porcine proximal tubule cells, were used. ATP (10(-6)M) or Ado (10(-6)M) specifically stimulated Na(+)-ATPase activity without any changes in (Na(+)+K(+))-ATPase activity. Our results show that the Ado effect is mediated by its conversion to ATP. Furthermore, it was observed that the effect of ATP was mimicked by UTP, ATPγS and 2-thio-UTP, an agonist of P2Y2 and P2Y4 receptors. In addition, ATP-stimulated Na(+)-ATPase activity involves protein kinase C (PKC). Our results indicate that ATP-induced stimulation of proximal tubule Na(+)-ATPase activity is mediated by a PKC-dependent P2Y2 and/or P2Y4 pathway. These findings provide new perspectives on the role of the effect of P2Y-mediated extracellular ATP on renal sodium handling. Copyright © 2013 Elsevier Inc. All rights reserved.

Changes in the F0F1-ATPase activity of irradiated Lactobacillus acidophilus in the presence of ceftazidime at low pH

International Nuclear Information System (INIS)

Kalantaryan, V.P.; Trchounian, A.H.; Soghomonyan, D.R.

2014-01-01

The aim of this study was the investigation of the effects of low intensity electromagnetic irradiation (EMI) at the frequencies of 51.8 and 53 GHz and of antibiotic ceftazidime on the N,N'-dicyclohexylcarbodiimide (DCCD) inhibited ATPase activity of membrane vesicles of lactic acid bacteria Lactobacillus acidophilus grown at low pH (pH 4.0 or 6.5) and assayed at the same pH. It was shown that both frequencies EMI stimulated ATPase activity of L. acidophilus grown at pH 4.0, but EMI combined with ceftazidime and DCCD decreased ATPase activity at pH 4.0 and pH 6.5. It was suggested that the F 0 F 1 -ATPase might be a target for EMI even at low pH

Cell Signaling Associated with Na+/K+-ATPase: Activation of Phosphatidylinositide 3-Kinase IA/Akt by Ouabain Is Independent of Src

Science.gov (United States)

2013-01-01

Exposure of intact cells to selective inhibitors of Na+/K+-ATPase such as ouabain activates several growth-related cell signaling pathways. It has been suggested that the initial event of these pathways is the binding of ouabain to a preexisting complex of Src with Na+/K+-ATPase of the plasma membrane. The aim of this work was to evaluate the role of Src in the ouabain-induced activation of phosphatidylinositide 3-kinase 1A (PI3K1A) and its downstream consequences. When fibroblasts devoid of Src (SYF cells) and controls (Src++ cells) were exposed to ouabain, PI3K1A, Akt, and proliferative growth were similarly stimulated in both cell lines. Ouabain-induced activation of Akt was not prevented by the Src inhibitor PP2. In contrast, ERK1/2 were not activated by ouabain in SYF cells but were stimulated in Src++ cells; this was prevented by PP2. In isolated adult mouse cardiac myocytes, where ouabain induces hypertrophic growth, PP2 also did not prevent ouabain-induced activation of Akt and the resulting hypertrophy. Ouabain-induced increases in the levels of co-immunoprecipitation of the α-subunit of Na+/K+-ATPase with the p85 subunit of PI3K1A were noted in SYF cells, Src++ cells, and adult cardiac myocytes. In conjunction with previous findings, the results presented here indicate that (a) if there is a preformed complex of Src and Na+/K+-ATPase, it is irrelevant to ouabain-induced activation of the PI3K1A/Akt pathway through Na+/K+-ATPase and (b) a more likely, but not established, mechanism of linkage of Na+/K+-ATPase to PI3K1A is the ouabain-induced interaction of a proline-rich domain of the α-subunit of Na+/K+-ATPase with the SH3 domain of the p85 subunit of PI3K1A. PMID:24266852

Plasma membrane calcium ATPases and related disorders.

Science.gov (United States)

Giacomello, Marta; De Mario, Agnese; Scarlatti, Chiara; Primerano, Simona; Carafoli, Ernesto

2013-03-01

The plasma membrane Ca(2+) ATPases (PMCA pumps) cooperate with other transport systems in the plasma membrane and in the organelles in the regulation of cell Ca(2+). They have high Ca(2+) affinity and are thus the fine tuners of cytosolic Ca(2+). They belong to the superfamily of P-type ATPases: their four basic isoforms share the essential properties of the reaction cycle and the general membrane topography motif of 10 transmembrane domains and three large cytosolic units. However they also differ in other important properties, e.g., tissue distribution and regulatory mechanisms. Their chief regulator is calmodulin, that removes their C-terminal cytosolic tail from autoinhibitory binding sites next to the active site of the pump, restoring activity. The number of pump isoforms is increased to over 30 by alternative splicing of the transcripts at a N-terminal site (site A) and at site C within the C-terminal calmodulin binding domain: the splice variants are tissue specific and developmentally regulated. The importance of PMCAs in the maintenance of cellular Ca(2+) homeostasis is underlined by the disease phenotypes, genetic or acquired, caused by their malfunction. Non-genetic PMCA deficiencies have long been considered possible causative factors in disease conditions as important as cancer, hypertension, or neurodegeneration. Those of genetic origin are better characterized: some have now been discovered in humans as well. They concern all four PMCA isoforms, and range from cardiac dysfunctions, to deafness, to hypertension, to cerebellar ataxia. Copyright © 2012 Elsevier Ltd. All rights reserved.

Exploration of flexible phenylpropylurea scaffold as novel cardiac myosin activators for the treatment of systolic heart failure.

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Manickam, Manoj; Jalani, Hitesh B; Pillaiyar, Thanigaimalai; Sharma, Niti; Boggu, Pulla Reddy; Venkateswararao, Eeda; Lee, You-Jung; Jeon, Eun-Seok; Jung, Sang-Hun

2017-07-07

A series of flexible urea derivatives have been synthesized and demonstrated as selective cardiac myosin ATPase activator. Among them 1-phenethyl-3-(3-phenylpropyl)urea (1, cardiac myosin ATPase activation at 10 μM = 51.1%; FS = 18.90; EF = 12.15) and 1-benzyl-3-(3-phenylpropyl)urea (9, cardiac myosin ATPase activation = 53.3%; FS = 30.04; EF = 18.27) showed significant activity in vitro and in vivo. The change of phenyl ring with tetrahydropyran-4-yl moiety viz., 1-(3-phenylpropyl)-3-((tetrahydro-2H-pyran-4-yl)methyl)urea (14, cardiac myosin ATPase activation = 81.4%; FS = 20.50; EF = 13.10), and morpholine moiety viz., 1-(2-morpholinoethyl)-3-(3-phenylpropyl)urea (21, cardiac myosin ATPase activation = 44.0%; FS = 24.79; EF = 15.65), proved to be efficient to activate the cardiac myosin. The potent compounds 1, 9, 14 and 21 were found to be selective for cardiac myosin over skeletal and smooth myosins. Thus, these urea derivatives are potent scaffold to develop as a newer cardiac myosin activator for the treatment of systolic heart failure. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Identification of small-molecule inhibitors of Yersinia pestis Type III secretion system YscN ATPase.

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Wieslaw Swietnicki

Full Text Available Yersinia pestis is a gram negative zoonotic pathogen responsible for causing bubonic and pneumonic plague in humans. The pathogen uses a type III secretion system (T3SS to deliver virulence factors directly from bacterium into host mammalian cells. The system contains a single ATPase, YscN, necessary for delivery of virulence factors. In this work, we show that deletion of the catalytic domain of the yscN gene in Y. pestis CO92 attenuated the strain over three million-fold in the Swiss-Webster mouse model of bubonic plague. The result validates the YscN protein as a therapeutic target for plague. The catalytic domain of the YscN protein was made using recombinant methods and its ATPase activity was characterized in vitro. To identify candidate therapeutics, we tested computationally selected small molecules for inhibition of YscN ATPase activity. The best inhibitors had measured IC(50 values below 20 µM in an in vitro ATPase assay and were also found to inhibit the homologous BsaS protein from Burkholderia mallei animal-like T3SS at similar concentrations. Moreover, the compounds fully inhibited YopE secretion by attenuated Y. pestis in a bacterial cell culture and mammalian cells at µM concentrations. The data demonstrate the feasibility of targeting and inhibiting a critical protein transport ATPase of a bacterial virulence system. It is likely the same strategy could be applied to many other common human pathogens using type III secretion system, including enteropathogenic E. coli, Shigella flexneri, Salmonella typhimurium, and Burkholderia mallei/pseudomallei species.

Identification of small-molecule inhibitors of Yersinia pestis Type III secretion system YscN ATPase.

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Swietnicki, Wieslaw; Carmany, Daniel; Retford, Michael; Guelta, Mark; Dorsey, Russell; Bozue, Joel; Lee, Michael S; Olson, Mark A

2011-01-01

Yersinia pestis is a gram negative zoonotic pathogen responsible for causing bubonic and pneumonic plague in humans. The pathogen uses a type III secretion system (T3SS) to deliver virulence factors directly from bacterium into host mammalian cells. The system contains a single ATPase, YscN, necessary for delivery of virulence factors. In this work, we show that deletion of the catalytic domain of the yscN gene in Y. pestis CO92 attenuated the strain over three million-fold in the Swiss-Webster mouse model of bubonic plague. The result validates the YscN protein as a therapeutic target for plague. The catalytic domain of the YscN protein was made using recombinant methods and its ATPase activity was characterized in vitro. To identify candidate therapeutics, we tested computationally selected small molecules for inhibition of YscN ATPase activity. The best inhibitors had measured IC(50) values below 20 µM in an in vitro ATPase assay and were also found to inhibit the homologous BsaS protein from Burkholderia mallei animal-like T3SS at similar concentrations. Moreover, the compounds fully inhibited YopE secretion by attenuated Y. pestis in a bacterial cell culture and mammalian cells at µM concentrations. The data demonstrate the feasibility of targeting and inhibiting a critical protein transport ATPase of a bacterial virulence system. It is likely the same strategy could be applied to many other common human pathogens using type III secretion system, including enteropathogenic E. coli, Shigella flexneri, Salmonella typhimurium, and Burkholderia mallei/pseudomallei species.

Ubxd1 is a novel co-factor of the human p97 ATPase

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Madsen, Louise; Andersen, Katrine M; Prag, Søren

2008-01-01

The AAA ATPase complex known as p97 or VCP in mammals and Cdc48 in yeast is connected to a multitude of cellular pathways, including membrane fusion, protein folding, protein degradation and activation of membrane-bound transcription factors. The mechanism by which p97 participates in such a broad...

Conformational changes in the AAA ATPase p97–p47 adaptor complex

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Beuron, Fabienne; Dreveny, Ingrid; Yuan, Xuemei; Pye, Valerie E; Mckeown, Ciaran; Briggs, Louise C; Cliff, Matthew J; Kaneko, Yayoi; Wallis, Russell; Isaacson, Rivka L; Ladbury, John E; Matthews, Steve J; Kondo, Hisao; Zhang, Xiaodong; Freemont, Paul S

2006-01-01

The AAA+ATPase p97/VCP, helped by adaptor proteins, exerts its essential role in cellular events such as endoplasmic reticulum-associated protein degradation or the reassembly of Golgi, ER and the nuclear envelope after mitosis. Here, we report the three-dimensional cryo-electron microscopy structures at ∼20 Å resolution in two nucleotide states of the endogenous hexameric p97 in complex with a recombinant p47 trimer, one of the major p97 adaptor proteins involved in membrane fusion. Depending on the nucleotide state, we observe the p47 trimer to be in two distinct arrangements on top of the p97 hexamer. By combining the EM data with NMR and other biophysical measurements, we propose a model of ATP-dependent p97(N) domain motions that lead to a rearrangement of p47 domains, which could result in the disassembly of target protein complexes. PMID:16601695

Nitric oxide and Na,K-ATPase activity in rat skeletal muscle

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Juel, Carsten

2016-01-01

Aim: It has been suggested that nitric oxide (NO) stimulates the Na,K-ATPase in cardiac myocytes. Therefore, the aims of this study were to investigate whether NO increases Na,K-ATPase activity in skeletal muscle and, if that is the case, to identify the underlying mechanism. Method: The study used...... isolated rat muscle, muscle homogenates and purified membranes as model systems. Na,K-ATPase activity was quantified from phosphate release due to ATP hydrolysis. Results: Exposure to the NO donor spermine NONOate (10 μm) increased the maximal Na,K-ATPase activity by 27% in isolated glycolytic muscles...... activity was depressed by oxidized glutathione. Conclusion: NO and cGMP stimulate the Na,K-ATPase in glycolytic skeletal muscle. Direct S-nitrosylation and interference with S-glutathionylation seem to be excluded. In addition, phosphorylation of phospholemman at serine 68 is not involved. Most likely...

Advances in targeting the vacuolar proton-translocating ATPase (V-ATPase for anti-fungal therapy

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Summer R. Hayek

2014-01-01

Full Text Available Vacuolar proton-translocating ATPase (V-ATPase is a membrane-bound, multi-subunit enzyme that uses the energy of ATP hydrolysis to pump protons across membranes. V-ATPase activity is critical for pH homeostasis and organelle acidification as well as for generation of the membrane potential that drives secondary transporters and cellular metabolism. V-ATPase is highly conserved across species and is best characterized in the model fungus Saccharomyces cerevisiae (S. cerevisiae. However, recent studies in mammals have identified significant alterations from fungi, particularly in the isoform composition of the 14 subunits and in the regulation of complex disassembly. These differences could be exploited for selectivity between fungi and humans and highlight the potential for V-ATPase as an anti-fungal drug target. Candida albicans (C. albicans is a major human fungal pathogen and causes fatality in 35% of systemic infections, even with anti-fungal treatment. The pathogenicity of C. albicans correlates with environmental, vacuolar, and cytoplasmic pH regulation, and V-ATPase appears to play a fundamental role in each of these processes. Genetic loss of V-ATPase in pathogenic fungi leads to defective virulence, and a comprehensive picture of the mechanisms involved is emerging. Recent studies have explored the practical utility of V-ATPase as an anti-fungal drug target in C. albicans, including pharmacological inhibition, azole therapy, and targeting of downstream pathways. This overview will discuss these studies as well as hypothetical ways to target V-ATPase and novel high-throughput methods for use in future drug discovery screens.

Na+,K+-ATPase Na+ affinity in rat skeletal muscle fiber types

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Kristensen, Michael; Juel, Carsten

2010-01-01

Previous studies in expression systems have found different ion activation of the Na(+)/K(+)-ATPase isozymes, which suggest that different muscles have different ion affinities. The rate of ATP hydrolysis was used to quantify Na(+),K(+)-ATPase activity, and the Na(+) affinity of Na(+),K(+)-ATPase...

Cellular function and pathological role of ATP13A2 and related P-type transport ATPases in Parkinson's disease and other neurological disorders

DEFF Research Database (Denmark)

van Veen, Sarah; Sørensen, Danny M.; Holemans, Tine

2014-01-01

-type ATPases. We critically review the available data concerning the role of ATP13A2 in heavy metal transport and propose a possible alternative hypothesis that ATP13A2 might be a flippase. As a flippase, ATP13A2 may transport an organic molecule, such as a lipid or a peptide, from one membrane leaflet...

Antagonism of the Sodium-Potassium ATPase Impairs Chikungunya Virus Infection

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Alison W. Ashbrook

2016-05-01

Full Text Available Chikungunya virus (CHIKV is a reemerging alphavirus that has caused epidemics of fever, arthralgia, and rash worldwide. There are currently no licensed vaccines or antiviral therapies available for the prevention or treatment of CHIKV disease. We conducted a high-throughput, chemical compound screen that identified digoxin, a cardiac glycoside that blocks the sodium-potassium ATPase, as a potent inhibitor of CHIKV infection. Treatment of human cells with digoxin or a related cardiac glycoside, ouabain, resulted in a dose-dependent decrease in infection by CHIKV. Inhibition by digoxin was cell type-specific, as digoxin treatment of either murine or mosquito cells did not diminish CHIKV infection. Digoxin displayed antiviral activity against other alphaviruses, including Ross River virus and Sindbis virus, as well as mammalian reovirus and vesicular stomatitis virus. The digoxin-mediated block to CHIKV and reovirus infection occurred at one or more postentry steps, as digoxin inhibition was not bypassed by fusion of CHIKV at the plasma membrane or infection with cell surface-penetrating reovirus entry intermediates. Selection of digoxin-resistant CHIKV variants identified multiple mutations in the nonstructural proteins required for replication complex formation and synthesis of viral RNA. These data suggest a role for the sodium-potassium ATPase in promoting postentry steps of CHIKV replication and provide rationale for modulation of this pathway as a broad-spectrum antiviral strategy.

Effects of solubilization on the inhibition of the p-type ATPase from maize roots by N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline.

Science.gov (United States)

Brauer, D K; Gurriel, M; Tu, S I

1992-12-01

The biochemical events utilized by transport proteins to convert the chemical energy from the hydrolysis of ATP into an electro-chemical gradient are poorly understood. The inhibition of the plasma membrane ATPase from corn (Zea mays L.) roots by N-(ethoxycarbonyl)-2-ethoxy-1,2-dihydroquinoline (EEDQ) was compared to that of ATPase solubilized with N-tetradecyl-N,N-dimethyl-3-ammonio-1-propane-sulfonate (3-14) to provide insight into the minimal functional unit. The chromatographic behavior of the 3-14-solubilized ATPase activity during size exclusion chromatography and glycerol gradient centrifugation indicated that the solubilized enzyme was in a monomeric form. Both plasma membrane-bound and solubilized ATPase were inhibited by EEDQ in a time- and concentration-dependent manner consistent with a first-order reaction. When the log of the reciprocal of the half-time for inhibition was plotted as a function of the log of the EEDQ concentration, straight lines were obtained with slopes of approximately 0.5 and 1.0 for membrane-bound and 3-14-solubilized ATPase, respectively, indicating a change in the number of polypeptides per functional ATPase complex induced by solubilization with 3-14.

Photolabeling and radioligand binding of human erythrocyte NaK-ATPase with 125I-derivatives of cymarin and digitoxigenin

International Nuclear Information System (INIS)

Lowndes, J.M.

1988-01-01

NaK-ATPase is an enzyme which maintains Na + and K + gradients across the plasma membrane of eukaryotic cells, and is specifically inhibited by cardiac glycosides. The cardiac glycoside binding site is located primarily on the catalytic α subunit but the glycoprotein β and proteolipid-γ subunits may also contribute to the structure of the site. In order to label the cardiac glycoside binding site of human erythrocytes, four photoaffinity ligands with very high specific radioactivity were synthesized. The compounds, which are abbreviated [ 125 I]AISC, [ 125 I]AIPP-GluD, [ 125 I]AIPP-GalD and [ 125 I]IA-GalD, were all effective photolabels for NaK-ATPase as shown by ouabain-protectable, covalent labeling of the α, β, and proteolipid-γ subunits. In order to study the possible existence of a very high affinity binding site in erythrocyte NaK-ATPase, a carrier-free radioligand, [ 125 I]I-TASC, was synthesized; this compound had the same structure as [ 125 I]AISC except that a light-sensitive azide group was replaced with a hydroxyl group. Competitive binding assays with cymarin against 0.2 nM [ 125 I]I-TASC suggested two classes of erythrocyte binding sites. Scatchard analysis of direct [ 125 I]I-TASC binding indicated that the very high affinity, low capacity class of erythrocyte bindings sites had a K D of 54 pM and a B max of 23 fmol/mg protein

Sperm Na+, K+-ATPase α4 and plasma membrane Ca2+-ATPase (PMCA) 4 regulation in asthenozoospermia.

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Lestari, Silvia W; Miati, Dessy Noor; Seoharso, P; Sugiyanto, R; Pujianto, Dwi A

2017-10-01

Asthenozoospermia, which is characterized by reduced motility, is one of the etiologies of male infertility. Its biochemical and functional consequences include altered ATPase activity. This study investigated the activities of Na + , K + -ATPase and Ca 2+ -ATPase and the expression of Na + , K + -ATPase α4 and PMCA4 isoforms in human sperm of asthenozoospermic infertile men. Nineteen samples from asthenozoospermic infertile couples were examined in this study. Computerized-assisted semen analysis (CASA) was performed, and the enzyme activity was measured based on the ability of ATPase to release organic phosphate from ATP as a substrate. The Na + , K + -ATPase α4 and PMCA4 isoform expression levels were measured by western immunoblotting, whereas the protein distribution was examined by immunocytochemistry. This showed that the Na + , K + -ATPase activity and the Na + , K + -ATPase α4 isoform expression were lower in the asthenozoospermia group than in the normozoospermia group (8.688±1.161 versus 13.851±1.884 µmol Pi/mg protein/h, respectively; p>0.05). In contrast, the Ca 2+ -ATPase activity was significantly higher in the asthenozoospermia group than in the normozoospermia group (11.154±1.186 versus 2.725±0.545 µmol Pi/mg protein/h, respectively; p0.05). The altered ATPase activity and isoform expression in asthenozoospermia may impair sperm structure and function.

Specialized functional diversity and interactions of the Na,K-ATPase

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Igor I. Krivoi

2016-05-01

Full Text Available Na,K-ATPase is a protein ubiquitously expressed in the plasma membrane of all animal cells and vitally essential for their functions. A specialized functional diversity of the Na,K-ATPase isozymes is provided by molecular heterogeneity, distinct subcellular localizations and functional interactions with molecular environment. Studies over the last decades clearly demonstrated complex and isoform-specific reciprocal functional interactions between the Na,K-ATPase and neighboring proteins and lipids. These interactions are enabled by a spatially restricted ion homeostasis, direct protein-protein/lipid interactions and protein kinase signaling pathways. In addition to its ‘classical’ function in ion translocation, the Na,K-ATPase is now considered as one of the most important signaling molecules in neuronal, epithelial, skeletal, cardiac and vascular tissues. Accordingly, the Na,K-ATPase forms specialized sub-cellular multimolecular microdomains which act as receptors to circulating endogenous cardiotonic steroids triggering a number of signaling pathways. Changes in these endogenous cardiotonic steroid levels and initiated signaling responses have significant adaptive values for tissues and whole organisms under numerous physiological and pathophysiological conditions. This review discusses recent progress in the studies of functional interactions between the Na,K-ATPase and molecular microenvironment, the Na,K-ATPase-dependent signaling pathways and their significance for diversity of cell function.

Mutations in the P-Type Cation-Transporter ATPase 4, PfATP4, Mediate Resistance to Both Aminopyrazole and Spiroindolone Antimalarials

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2015-01-01

Aminopyrazoles are a new class of antimalarial compounds identified in a cellular antiparasitic screen with potent activity against Plasmodium falciparum asexual and sexual stage parasites. To investigate their unknown mechanism of action and thus identify their target, we cultured parasites in the presence of a representative member of the aminopyrazole series, GNF-Pf4492, to select for resistance. Whole genome sequencing of three resistant lines showed that each had acquired independent mutations in a P-type cation-transporter ATPase, PfATP4 (PF3D7_1211900), a protein implicated as the novel Plasmodium spp. target of another, structurally unrelated, class of antimalarials called the spiroindolones and characterized as an important sodium transporter of the cell. Similarly to the spiroindolones, GNF-Pf4492 blocks parasite transmission to mosquitoes and disrupts intracellular sodium homeostasis. Our data demonstrate that PfATP4 plays a critical role in cellular processes, can be inhibited by two distinct antimalarial pharmacophores, and supports the recent observations that PfATP4 is a critical antimalarial target. PMID:25322084

Subcellular distribution of calcium-binding proteins and a calcium-ATPase in canine pancreas

International Nuclear Information System (INIS)

Nigam, S.K.; Towers, T.

1990-01-01

Using a 45Ca blot-overlay assay, we monitored the subcellular fractionation pattern of several Ca binding proteins of apparent molecular masses 94, 61, and 59 kD. These proteins also appeared to stain blue with Stains-All. Additionally, using a monoclonal antiserum raised against canine cardiac sarcoplasmic reticulum Ca-ATPase, we examined the subcellular distribution of a canine pancreatic 110-kD protein recognized by this antiserum. This protein had the same electrophoretic mobility as the cardiac protein against which the antiserum was raised. The three Ca binding proteins and the Ca-ATPase cofractionated into the rough microsomal fraction (RM), previously shown to consist of highly purified RER, in a pattern highly similar to that of the RER marker, ribophorin I. To provide further evidence for an RER localization, native RM were subjected to isopycnic flotation in sucrose gradients. The Ca binding proteins and the Ca-ATPase were found in dense fractions, along with ribophorin I. When RM were stripped of ribosomes with puromycin/high salt, the Ca binding proteins and the Ca-ATPase exhibited a shift to less dense fractions, as did ribophorin I. We conclude that, in pancreas, the Ca binding proteins and Ca-ATPase we detect are localized to the RER (conceivably a subcompartment of the RER) or, possibly, a structure intimately associated with the RER

Characterization of the plasma membrane H+-ATPase in the liverwort Marchantia polymorpha.

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Okumura, Masaki; Inoue, Shin-ichiro; Takahashi, Koji; Ishizaki, Kimitsune; Kohchi, Takayuki; Kinoshita, Toshinori

2012-06-01

The plasma membrane H(+)-ATPase generates an electrochemical gradient of H(+) across the plasma membrane that provides the driving force for solute transport and regulates pH homeostasis and membrane potential in plant cells. Recent studies have demonstrated that phosphorylation of the penultimate threonine in H(+)-ATPase and subsequent binding of a 14-3-3 protein is the major common activation mechanism for H(+)-ATPase in vascular plants. However, there is very little information on the plasma membrane H(+)-ATPase in nonvascular plant bryophytes. Here, we show that the liverwort Marchantia polymorpha, which is the most basal lineage of extant land plants, expresses both the penultimate threonine-containing H(+)-ATPase (pT H(+)-ATPase) and non-penultimate threonine-containing H(+)-ATPase (non-pT H(+)-ATPase) as in the green algae and that pT H(+)-ATPase is regulated by phosphorylation of its penultimate threonine. A search in the expressed sequence tag database of M. polymorpha revealed eight H(+)-ATPase genes, designated MpHA (for M. polymorpha H(+)-ATPase). Four isoforms are the pT H(+)-ATPase; the remaining isoforms are non-pT H(+)-ATPase. An apparent 95-kD protein was recognized by anti-H(+)-ATPase antibodies against an Arabidopsis (Arabidopsis thaliana) isoform and was phosphorylated on the penultimate threonine in response to the fungal toxin fusicoccin in thalli, indicating that the 95-kD protein contains pT H(+)-ATPase. Furthermore, we found that the pT H(+)-ATPase in thalli is phosphorylated in response to light, sucrose, and osmotic shock and that light-induced phosphorylation depends on photosynthesis. Our results define physiological signals for the regulation of pT H(+)-ATPase in the liverwort M. polymorpha, which is one of the earliest plants to acquire pT H(+)-ATPase.

Deinhibition of cardiac Na/sup +/-K/sup +/-ATPase after exposure to exogenous phospholipase A/sub 2/

Energy Technology Data Exchange (ETDEWEB)

Colvin, R.A.

1987-01-01

After 2 h of exogenous phospholipase A/sub 2/ (PLA/sub 2/) exposure, membrane phospholipid decreased from 3.22 +/- 0.31 to 1.06 +/- 0.13 ..mu..mol/mg (33% of control). All classes of phospholipid, except sphingomyelin, were hydrolyzed, whereas total cholesterol content was unaffected. Increases in nonesterified fatty acids (NEFA) were reflected primarily in oleic (18:1), linoleic (18:2), and arachidonic (20:4). Na/sup +/-K/sup +/-adenosinetriphosphatase (ATPase) activity was inhibited to 29% of control by 2 h of PLA/sub 2/ treatment, and this inhibition was reversed (albeit, not completely after 5 min of PLA/sub 2/ treatment) by removal of the hydrolysis products with 0.1% bovine serum albumin (BSA). In contrast, the apparent binding capacity for (/sup 3/H)ouabain was not affected by PLA/sub 2/ treatment. Unmasking of latent (/sup 3/H)ouabain binding by alamethicin was utilized to estimate changes in the proportion of sealed vesicles present before and after PLA/sub 2/ treatment. PLA/sub 2/ treatment resulted in a time-dependent loss of sealed vesicles that paralleled the time course of phospholipid hydrolysis and was not reversed by washing with BSA. These studies demonstrate that cardiac Na/sup +/-K/sup +/-ATPase activity is inhibited by accumulation of endogenously produced lysophospholipids and NEFA. In contrast, loss of vesicle integrity may result from both accumulation of endogenously produced hydrolysis products and membrane phospholipid depletion.

Exercise and type 2 diabetes mellitus: changes in tissue-specific fat distribution and cardiac function.

Science.gov (United States)

Jonker, Jacqueline T; de Mol, Pieter; de Vries, Suzanna T; Widya, Ralph L; Hammer, Sebastiaan; van Schinkel, Linda D; van der Meer, Rutger W; Gans, Rijk O B; Webb, Andrew G; Kan, Hermien E; de Koning, Eelco J P; Bilo, Henk J G; Lamb, Hildo J

2013-11-01

To prospectively assess the effects of an exercise intervention on organ-specific fat accumulation and cardiac function in type 2 diabetes mellitus. Written informed consent was obtained from all participants, and the study protocol was approved by the medical ethics committee. The study followed 12 patients with type 2 diabetes mellitus (seven men; mean age, 46 years ± 2 [standard error]) before and after 6 months of moderate-intensity exercise, followed by a high-altitude trekking expedition with exercise of long duration. Abdominal, epicardial, and paracardial fat volume were measured by using magnetic resonance (MR) imaging. Cardiac function was quantified with cardiac MR, and images were analyzed by a researcher who was supervised by a senior researcher (4 and 21 years of respective experience in cardiac MR). Hepatic, myocardial, and intramyocellular triglyceride (TG) content relative to water were measured with proton MR spectroscopy at 1.5 and 7 T. Two-tailed paired t tests were used for statistical analysis. Exercise reduced visceral abdominal fat volume from 348 mL ± 57 to 219 mL ± 33 (P Exercise decreased hepatic TG content from 6.8% ± 2.3 to 4.6% ± 1.6 (P Exercise did not change epicardial fat volume (P = .9), myocardial TG content (P = .9), intramyocellular lipid content (P = .3), or cardiac function (P = .5). A 6-month exercise intervention in type 2 diabetes mellitus decreased hepatic TG content and visceral abdominal and paracardial fat volume, which are associated with increased cardiovascular risk, but cardiac function was unaffected. Tissue-specific exercise-induced changes in body fat distribution in type 2 diabetes mellitus were demonstrated in this study. RSNA, 2013

The plant P4-ATPase ALA2 is involved in flipping of phosphatidylserine analogues

DEFF Research Database (Denmark)

Lopez Marques, Rosa Laura

The plant P4-ATPase ALA2 is involved in flipping of phosphatidylserine analogues Rosa Laura López-Marqués1, Lisbeth Rosager Poulsen1, Katharina Meffert2, Thomas Pomorski2, Michael Gjedde Palmgren1 1Centre for Membrane Pumps in Cells and Disease - PUMPKIN, Danish National Research Foundation...... physiological function.   1 Poulsen, L.R; López-Marqués, R.L et al. (2008) The Arabidopsis P4-ATPase ALA3 localizes to the Golgi and requires a ß-subunit to function in lipid translocation and secretory vesicle formation. The Plant Cell, vol. 20, 658-676. 2 Gomès, E. et al. (2000) Chilling tolerance...... in Arabidopsis involves ALA1, a member of a new family of putative aminophospholipid translocases. The Plant Cell, vol. 12, 2441-2453....

The plant P4-ATPase ALA2 is involved in flipping of phosphatidylserine analogues

DEFF Research Database (Denmark)

Poulsen, Lisbeth Rosager

  The plant P4-ATPase ALA2 is involved in flipping of phosphatidylserine analogues Rosa Laura López-Marqués1, Lisbeth Rosager Poulsen1, Katharina Meffert2, Thomas Pomorski2, Michael Gjedde Palmgren1 1Centre for Membrane Pumps in Cells and Disease - PUMPKIN, Danish National Research Foundation...... physiological function.   1 Poulsen, L.R; López-Marqués, R.L et al. (2008) The Arabidopsis P4-ATPase ALA3 localizes to the Golgi and requires a ß-subunit to function in lipid translocation and secretory vesicle formation. The Plant Cell, vol. 20, 658-676. 2 Gomès, E. et al. (2000) Chilling tolerance...... in Arabidopsis involves ALA1, a member of a new family of putative aminophospholipid translocases. The Plant Cell, vol. 12, 2441-2453....

The epigenetic regulator Smchd1 contains a functional GHKL-type ATPase domain.

Science.gov (United States)

Chen, Kelan; Dobson, Renwick C J; Lucet, Isabelle S; Young, Samuel N; Pearce, F Grant; Blewitt, Marnie E; Murphy, James M

2016-06-15

Structural maintenance of chromosomes flexible hinge domain containing 1 (Smchd1) is an epigenetic regulator that plays critical roles in gene regulation during development. Mutations in SMCHD1 were recently implicated in the pathogenesis of facioscapulohumeral muscular dystrophy (FSHD), although the mechanistic basis remains of outstanding interest. We have previously shown that Smchd1 associates with chromatin via its homodimeric C-terminal hinge domain, yet little is known about the function of the putative GHKL (gyrase, Hsp90, histidine kinase, MutL)-type ATPase domain at its N-terminus. To formally assess the structure and function of Smchd1's ATPase domain, we have generated recombinant proteins encompassing the predicted ATPase domain and the adjacent region. Here, we show that the Smchd1 N-terminal region exists as a monomer and adopts a conformation resembling that of monomeric full-length heat shock protein 90 (Hsp90) protein in solution, even though the two proteins share only ∼8% overall sequence identity. Despite being monomeric, the N-terminal region of Smchd1 exhibits ATPase activity, which can be antagonized by the reaction product, ADP, or the Hsp90 inhibitor, radicicol, at a nanomolar concentration. Interestingly, introduction of an analogous mutation to that identified in SMCHD1 of an FSHD patient compromised protein stability, suggesting a possible molecular basis for loss of protein function and pathogenesis. Together, these results reveal important structure-function characteristics of Smchd1 that may underpin its mechanistic action at the chromatin level. © 2016 The Author(s). published by Portland Press Limited on behalf of the Biochemical Society.

Ataxia telangiectasia mutated (ATM) interacts with p400 ATPase for an efficient DNA damage response.

Science.gov (United States)

Smith, Rebecca J; Savoian, Matthew S; Weber, Lauren E; Park, Jeong Hyeon

2016-11-04

Ataxia telangiectasia mutated (ATM) and TRRAP proteins belong to the phosphatidylinositol 3-kinase-related kinase family and are involved in DNA damage repair and chromatin remodeling. ATM is a checkpoint kinase that is recruited to sites of DNA double-strand breaks where it phosphorylates a diverse range of proteins that are part of the chromatin and DNA repair machinery. As an integral subunit of the TRRAP-TIP60 complexes, p400 ATPase is a chromatin remodeler that is also targeted to DNA double-strand break sites. While it is understood that DNA binding transcriptional activators recruit p400 ATPase into a regulatory region of the promoter, how p400 recognises and moves to DNA double-strand break sites is far less clear. Here we investigate a possibility whether ATM serves as a shuttle to deliver p400 to break sites. Our data indicate that p400 co-immunoprecipitates with ATM independently of DNA damage state and that the N-terminal domain of p400 is vital for this interaction. Heterologous expression studies using Sf9 cells revealed that the ATM-p400 complex can be reconstituted without other mammalian bridging proteins. Overexpression of ATM-interacting p400 regions in U2OS cells induced dominant negative effects including the inhibition of both DNA damage repair and cell proliferation. Consistent with the dominant negative effect, the stable expression of an N-terminal p400 fragment showed a decrease in the association of p400 with ATM, but did not alter the association of p400 with TRRAP. Taken together, our findings suggest that a protein-protein interaction between ATM and p400 ATPase occurs independently of DNA damage and contributes to efficient DNA damage response and repair.

Structure and Function of p97 and Pex1/6 Type II AAA+ Complexes.

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Saffert, Paul; Enenkel, Cordula; Wendler, Petra

2017-01-01

Protein complexes of the Type II AAA+ (ATPases associated with diverse cellular activities) family are typically hexamers of 80-150 kDa protomers that harbor two AAA+ ATPase domains. They form double ring assemblies flanked by associated domains, which can be N-terminal, intercalated or C-terminal to the ATPase domains. Most prominent members of this family include NSF (N-ethyl-maleimide sensitive factor), p97/VCP (valosin-containing protein), the Pex1/Pex6 complex and Hsp104 in eukaryotes and ClpB in bacteria. Tremendous efforts have been undertaken to understand the conformational dynamics of protein remodeling type II AAA+ complexes. A uniform mode of action has not been derived from these works. This review focuses on p97/VCP and the Pex1/6 complex, which both structurally remodel ubiquitinated substrate proteins. P97/VCP plays a role in many processes, including ER- associated protein degradation, and the Pex1/Pex6 complex dislocates and recycles the transport receptor Pex5 from the peroxisomal membrane during peroxisomal protein import. We give an introduction into existing knowledge about the biochemical and cellular activities of the complexes before discussing structural information. We particularly emphasize recent electron microscopy structures of the two AAA+ complexes and summarize their structural differences.

Distinct pH dependencies of Na+/K+ selectivity at the two faces of Na,K-ATPase.

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Cornelius, Flemming; Tsunekawa, Naoki; Toyoshima, Chikashi

2018-02-09

The sodium pump (Na,K-ATPase) in animal cells is vital for actively maintaining ATP hydrolysis-powered Na + and K + electrochemical gradients across the cell membrane. These ion gradients drive co- and countertransport and are critical for establishing the membrane potential. It has been an enigma how Na,K-ATPase discriminates between Na + and K + , despite the pumped ion on each side being at a lower concentration than the other ion. Recent crystal structures of analogs of the intermediate conformations E2·Pi·2K + and Na + -bound E1∼P·ADP suggest that the dimensions of the respective binding sites in Na,K-ATPase are crucial in determining its selectivity. Here, we found that the selectivity at each membrane face is pH-dependent and that this dependence is unique for each face. Most notable was a strong increase in the specific affinity for K + at the extracellular face ( i.e. E2 conformation) as the pH is lowered from 7.5 to 5. We also observed a smaller increase in affinity for K + on the cytoplasmic side (E1 conformation), which reduced the selectivity for Na + Theoretical analysis of the p K a values of ion-coordinating acidic amino acid residues suggested that the face-specific pH dependences and Na + /K + selectivities may arise from the protonation or ionization of key residues. The increase in K + selectivity at low pH on the cytoplasmic face, for instance, appeared to be associated with Asp 808 protonation. We conclude that changes in the ionization state of coordinating residues in Na,K-ATPase could contribute to altering face-specific ion selectivity. © 2018 by The American Society for Biochemistry and Molecular Biology, Inc.

Standardization of metachromatic staining method of myofibrillar ATPase activity of myosin to skeletal striated muscle of mules and donkeys

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Flora H.F. D'Angelis

2014-09-01

Full Text Available This study aims at standardizing the pre-incubation and incubation pH and temperature used in the metachromatic staining method of myofibrillar ATPase activity of myosin (mATPase used for asses and mules. Twenty four donkeys and 10 mules, seven females and three males, were used in the study. From each animal, fragments from the Gluteus medius muscle were collected and percutaneous muscle biopsy was performed using a 6.0-mm Bergström-type needle. In addition to the metachromatic staining method of mATPase, the technique of nicotinamide adenine dinucleotide tetrazolium reductase (NADH-TR was also performed to confirm the histochemical data. The histochemical result of mATPase for acidic pre-incubation (pH=4.50 and alkaline incubation (pH=10.50, at a temperature of 37ºC, yielded the best differentiation of fibers stained with toluidine blue. Muscle fibers were identified according to the following colors: type I (oxidative, light blue, type IIA (oxidative-glycolytic, intermediate blue and type IIX (glycolytic, dark blue. There are no reports in the literature regarding the characterization and distribution of different types of muscle fibers used by donkeys and mules when performing traction work, cargo transportation, endurance sports (horseback riding and marching competitions. Therefore, this study is the first report on the standardization of the mATPase technique for donkeys and mules.

Structural Features Reminiscent of ATP-Driven Protein Translocases Are Essential for the Function of a Type III Secretion-Associated ATPase.

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Kato, Junya; Lefebre, Matthew; Galán, Jorge E

2015-09-01

Many bacterial pathogens and symbionts utilize type III secretion systems to interact with their hosts. These machines have evolved to deliver bacterial effector proteins into eukaryotic target cells to modulate a variety of cellular functions. One of the most conserved components of these systems is an ATPase, which plays an essential role in the recognition and unfolding of proteins destined for secretion by the type III pathway. Here we show that structural features reminiscent of other ATP-driven protein translocases are essential for the function of InvC, the ATPase associated with a Salmonella enterica serovar Typhimurium type III secretion system. Mutational and functional analyses showed that a two-helix-finger motif and a conserved loop located at the entrance of and within the predicted pore formed by the hexameric ATPase are essential for InvC function. These findings provide mechanistic insight into the function of this highly conserved component of type III secretion machines. Type III secretion machines are essential for the virulence or symbiotic relationships of many bacteria. These machines have evolved to deliver bacterial effector proteins into host cells to modulate cellular functions, thus facilitating bacterial colonization and replication. An essential component of these machines is a highly conserved ATPase, which is necessary for the recognition and secretion of proteins destined to be delivered by the type III secretion pathway. Using modeling and structure and function analyses, we have identified structural features of one of these ATPases from Salmonella enterica serovar Typhimurium that help to explain important aspects of its function. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

E-1020, a water soluble imidazopyridine, has direct effects on Ca(2+)-dependent force and ATP hydrolysis of canine and bovine cardiac myofilaments.

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Powers, F M; Palmiter, K A; Solaro, R J

1996-01-01

E-1020 is a cardiotonic agent that acts as a cyclic-AMP phosphodiesterase inhibitor but also may have actions which alter myofilament response to Ca2+. To identify direct actions of E-1020 on cardiac contractile proteins, effects of E-1020 on myofibrillar Ca2+ dependent MgATPase and force generation in chemically skinned fiber bundles were measured. In bovine cardiac myofibrils, E-1020 (100 microM) significantly increased myofilament Ca2+ sensitivity and Ca(2+)-dependent ATPase activity at submaximal pCa values. At pCa 6.75, E-1020 significantly increased ATPase activity in bovine (10-100 microM) and canine (1-100 microM) cardiac myofibrils but had no effect on rat cardiac myofibrils. Moreover, in one population of canine ventricular fiber bundles, E-1020 (0.01-10 microM) significantly increased isometric tension at pCa 6.5 and 6.0, whereas in another population of bundles E-1020 had no effect on tension. In no case was resting (pCa 8.0) or maximal tension (pCa 4.5) increased by E-1020. Measurements of Ca2+ binding to canine ventricular skinned fiber preparations demonstrated that E-1020 does not alter the affinity of myofilament troponin C for Ca2+. We conclude that part of the mechanism by which E-1020 acts as an inotropic agent may involve alterations in the responsiveness of contractile proteins to Ca2+. The lack of effect of E-1020 on some preparations may be dependent on isoform populations of myofilament proteins.

Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart

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Habeck, Michael; Tokhtaeva, Elmira; Nadav, Yotam; Ben Zeev, Efrat; Ferris, Sean P.; Kaufman, Randal J.; Bab-Dinitz, Elizabeta; Kaplan, Jack H.; Dada, Laura A.; Farfel, Zvi; Tal, Daniel M.; Katz, Adriana; Sachs, George; Vagin, Olga; Karlish, Steven J. D.

2016-01-01

The Na,K-ATPase α2 subunit plays a key role in cardiac muscle contraction by regulating intracellular Ca2+, whereas α1 has a more conventional role of maintaining ion homeostasis. The β subunit differentially regulates maturation, trafficking, and activity of α-β heterodimers. It is not known whether the distinct role of α2 in the heart is related to selective assembly with a particular one of the three β isoforms. We show here by immunofluorescence and co-immunoprecipitation that α2 is preferentially expressed with β2 in T-tubules of cardiac myocytes, forming α2β2 heterodimers. We have expressed human α1β1, α2β1, α2β2, and α2β3 in Pichia pastoris, purified the complexes, and compared their functional properties. α2β2 and α2β3 differ significantly from both α2β1 and α1β1 in having a higher K0.5K+ and lower K0.5Na+ for activating Na,K-ATPase. These features are the result of a large reduction in binding affinity for extracellular K+ and shift of the E1P-E2P conformational equilibrium toward E1P. A screen of perhydro-1,4-oxazepine derivatives of digoxin identified several derivatives (e.g. cyclobutyl) with strongly increased selectivity for inhibition of α2β2 and α2β3 over α1β1 (range 22–33-fold). Molecular modeling suggests a possible basis for isoform selectivity. The preferential assembly, specific T-tubular localization, and low K+ affinity of α2β2 could allow an acute response to raised ambient K+ concentrations in physiological conditions and explain the importance of α2β2 for cardiac muscle contractility. The high sensitivity of α2β2 to digoxin derivatives explains beneficial effects of cardiac glycosides for treatment of heart failure and potential of α2β2-selective digoxin derivatives for reducing cardiotoxicity. PMID:27624940

A structural view on the functional importance of the sugar moiety and steroid hydroxyls of cardiotonic steroids in binding to Na,K-ATPase.

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Cornelius, Flemming; Kanai, Ryuta; Toyoshima, Chikashi

2013-03-01

The Na,K-ATPase is specifically inhibited by cardiotonic steroids (CTSs) like digoxin and is of significant therapeutic value in the treatment of congestive heart failure and arrhythmia. Recently, new interest has arisen in developing Na,K-ATPase inhibitors as anticancer agents. In the present study, we compare the potency and rate of inhibition as well as the reactivation of enzyme activity following inhibition by various cardiac glycosides and their aglycones at different pH values using shark Na,K-ATPase stabilized in the E2MgPi or in the E2BeFx conformations. The effects of the number and nature of various sugar residues as well as changes in the positions of hydroxyl groups on the β-side of the steroid core of cardiotonic steroids were investigated by comparing various cardiac glycoside compounds like ouabain, digoxin, digitoxin, and gitoxin with their aglycones. The results confirm our previous hypothesis that CTS binds primarily to the E2-P ground state through an extracellular access channel and that binding of extracellular Na(+) ions to K(+) binding sites relieved the CTS inhibition. This reactivation depended on the presence or absence of the sugar moiety on the CTS, and a single sugar is enough to impede reactivation. Finally, increasing the number of hydroxyl groups of the steroid was sterically unfavorable and was found to decrease the inhibitory potency and to confer high pH sensitivity, depending on their position on the steroid β-face. The results are discussed with reference to the recent crystal structures of Na,K-ATPase in the unbound and ouabain-bound states.

The yeast protein kinase Sch9 adjusts V-ATPase assembly/disassembly to control pH homeostasis and longevity in response to glucose availability.

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Tobias Wilms

2017-06-01

Full Text Available The conserved protein kinase Sch9 is a central player in the nutrient-induced signaling network in yeast, although only few of its direct substrates are known. We now provide evidence that Sch9 controls the vacuolar proton pump (V-ATPase to maintain cellular pH homeostasis and ageing. A synthetic sick phenotype arises when deletion of SCH9 is combined with a dysfunctional V-ATPase, and the lack of Sch9 has a significant impact on cytosolic pH (pHc homeostasis. Sch9 physically interacts with, and influences glucose-dependent assembly/disassembly of the V-ATPase, thereby integrating input from TORC1. Moreover, we show that the role of Sch9 in regulating ageing is tightly connected with V-ATPase activity and vacuolar acidity. As both Sch9 and the V-ATPase are highly conserved in higher eukaryotes, it will be interesting to further clarify their cooperative action on the cellular processes that influence growth and ageing.

Metal fluoride complexes of Na,K-ATPase: characterization of fluoride-stabilized phosphoenzyme analogues and their interaction with cardiotonic steroids.

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Cornelius, Flemming; Mahmmoud, Yasser A; Toyoshima, Chikashi

2011-08-26

The Na,K-ATPase belongs to the P-type ATPase family of primary active cation pumps. Metal fluorides like magnesium-, beryllium-, and aluminum fluoride act as phosphate analogues and inhibit P-type ATPases by interacting with the phosphorylation site, stabilizing conformations that are analogous to specific phosphoenzyme intermediates. Cardiotonic steroids like ouabain used in the treatment of congestive heart failure and arrhythmias specifically inhibit the Na,K-ATPase, and the detailed structure of the highly conserved binding site has recently been described by the crystal structure of the shark Na,K-ATPase in a state analogous to E2·2K(+)·P(i) with ouabain bound with apparently low affinity (1). In the present work inhibition, and subsequent reactivation by high Na(+), after treatment of shark Na,K-ATPase with various metal fluorides are characterized. Half-maximal inhibition of Na,K-ATPase activity by metal fluorides is in the micromolar range. The binding of cardiotonic steroids to the metal fluoride-stabilized enzyme forms was investigated using the fluorescent ouabain derivative 9-anthroyl ouabain and compared with binding to phosphorylated enzyme. The fastest binding was to the Be-fluoride stabilized enzyme suggesting a preformed ouabain binding cavity, in accord with results for Ca-ATPase where Be-fluoride stabilizes the E2-P ground state with an open luminal ion access pathway, which in Na,K-ATPase could be a passage for ouabain. The Be-fluoride stabilized enzyme conformation closely resembles the E2-P ground state according to proteinase K cleavage. Ouabain, but not its aglycone ouabagenin, prevented reactivation of this metal fluoride form by high Na(+) demonstrating the pivotal role of the sugar moiety in closing the extracellular cation pathway.

Regulation of vacuolar H+-ATPase in microglia by RANKL

International Nuclear Information System (INIS)

Serrano, Eric M.; Ricofort, Ryan D.; Zuo, Jian; Ochotny, Noelle; Manolson, Morris F.; Holliday, L. Shannon

2009-01-01

Vacuolar H + -ATPases (V-ATPases) are large electrogenic proton pumps composed of numerous subunits that play vital housekeeping roles in the acidification of compartments of the endocytic pathway. Additionally, V-ATPases play specialized roles in certain cell types, a capacity that is linked to cell type selective expression of isoforms of some of the subunits. We detected low levels of the a3 isoform of the a-subunit in mouse brain extracts. Examination of various brain-derived cell types by immunoblotting showed a3 was expressed in the N9 microglia cell line and in primary microglia, but not in other cell types. The expression of a3 in osteoclasts requires stimulation by Receptor Activator of Nuclear Factor κB-ligand (RANKL). We found that Receptor Activator of Nuclear Factor κB (RANK) was expressed by microglia. Stimulation of microglia with RANKL triggered increased expression of a3. V-ATPases in microglia were shown to bind microfilaments, and stimulation with RANKL increased the proportion of V-ATPase associated with the detergent-insoluble cytoskeletal fraction and with actin. In summary, microglia express the a3-subunit of V-ATPase. The expression of a3 and the interaction between V-ATPases and microfilaments was modulated by RANKL. These data suggest a novel molecular pathway for regulating microglia.

Reconstruction of the complete ouabain-binding pocket of Na,K-ATPase in gastric H,K-ATPase by substitution of only seven amino acids

NARCIS (Netherlands)

Qiu, L.Y.; Krieger, E.; Schaftenaar, G.; Swarts, H.G.P.; Willems, P.H.G.M.; Pont, J.J.H.H.M. de; Koenderink, J.B.

2005-01-01

Although cardiac glycosides have been used as drugs for more than 2 centuries and their primary target, the sodium pump (Na, K-ATPase), has already been known for 4 decades, their exact binding site is still elusive. In our efforts to define the molecular basis of digitalis glycosides binding we

Reconstruction of the complete ouabain-binding pocket of Na,K-ATPase in gastric H,K-ATPase by substitution of only seven amino acids.

NARCIS (Netherlands)

Qiu, L.; Krieger, E.; Schaftenaar, G.; Swarts, H.G.P.; Willems, P.H.G.M.; Pont, J.J.H.H.M. de; Koenderink, J.B.

2005-01-01

Although cardiac glycosides have been used as drugs for more than 2 centuries and their primary target, the sodium pump (Na,K-ATPase), has already been known for 4 decades, their exact binding site is still elusive. In our efforts to define the molecular basis of digitalis glycosides binding we

Steroid-like compounds in Chinese medicines promote blood circulation via inhibition of Na+/K+ -ATPase.

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Chen, Ronald J Y; Chung, Tse-yu; Li, Feng-yin; Yang, Wei-hung; Jinn, Tzyy-rong; Tzen, Jason T C

2010-06-01

To examine if steroid-like compounds found in many Chinese medicinal products conventionally used for the promotion of blood circulation may act as active components via the same molecular mechanism triggered by cardiac glycosides, such as ouabain. The inhibitory potency of ouabain and the identified steroid-like compounds on Na(+)/K(+)-ATPase activity was examined and compared. Molecular modeling was exhibited for the docking of these compounds to Na(+)/K(+)-ATPase. All the examined steroid-like compounds displayed more or less inhibition on Na(+)/K(+)-ATPase, with bufalin (structurally almost equivalent to ouabain) exhibiting significantly higher inhibitory potency than the others. In the pentacyclic triterpenoids examined, ursolic acid and oleanolic acid were moderate inhibitors of Na(+)/K(+)-ATPase, and their inhibitory potency was comparable to that of ginsenoside Rh2. The relatively high inhibitory potency of ursolic acid or oleanolic acid was due to the formation of a hydrogen bond between its carboxyl group and the Ile322 residue in the deep cavity close to two K(+) binding sites of Na(+)/K(+)-ATPase. Moreover, the drastic difference observed in the inhibitory potency of ouabain, bufalin, ginsenoside Rh2, and pentacyclic triterpenoids is ascribed mainly to the number of hydrogen bonds and partially to the strength of hydrophobic interaction between the compounds and residues around the deep cavity of Na(+)/K(+)-ATPase. Steroid-like compounds seem to contribute to therapeutic effects of many cardioactive Chinese medicinal products. Chinese herbs, such as Prunella vulgaris L, rich in ursolic acid, oleanolic acid and their glycoside derivatives may be adequate sources for cardiac therapy via effective inhibition on Na(+)/K(+)-ATPase.

Single mutation confers vanadate resistance to the plasma membrane H+-ATPase from the yeast Schizosaccharomyces pombe

International Nuclear Information System (INIS)

Ulaszewski, S.; Van Herck, J.C.; Dufour, J.P.; Kulpa, J.; Nieuwenhuis, B.; Goffeau, A.

1987-01-01

A single-gene nuclear mutant has been selected from the yeast Schizosaccharomyces pombe for growth resistance to Dio-9, a plasma membrane H+-ATPase inhibitor. From this mutant, called pma1, an ATPase activity has been purified. It contains a Mr = 100,000 major polypeptide which is phosphorylated by [gamma- 32 P] ATP. Proton pumping is not impaired since the isolated mutant ATPase is able, in reconstituted proteoliposomes, to quench the fluorescence of the delta pH probe 9-amino-6-chloro-2-methoxy acridine. The isolated mutant ATPase is sensitive to Dio-9 as well as to seven other plasma membrane H+-ATPase inhibitors. The mutant H+-ATPase activity tested in vitro is, however, insensitive to vanadate. Its Km for MgATP is modified and its ATPase specific activity is decreased. The pma1 mutation decreases the rate of extracellular acidification induced by glucose when cells are incubated at pH 4.5 under nongrowing conditions. During growth, the intracellular mutant pH is more acid than the wild type one. The derepression by ammonia starvation of methionine transport is decreased in the mutant. The growth rate of pma1 mutants is reduced in minimal medium compared to rich medium, especially when combined to an auxotrophic mutation. It is concluded that the H+-ATPase activity from yeast plasma membranes controls the intracellular pH as well as the derepression of amino acid, purine, and pyrimidine uptakes. The pma1 mutation modifies several transport properties of the cells including those responsible for the uptake of Dio-9 and other inhibitors

Cation Transport Coupled to ATP Hydrolysis by the (Na, K)-ATPase: An Integrated, Animated Model

Science.gov (United States)

Leone, Francisco A.; Furriel, Rosa P. M.; McNamara, John C.; Horisberger, Jean D.; Borin, Ivana A.

2010-01-01

An Adobe[R] animation is presented for use in undergraduate Biochemistry courses, illustrating the mechanism of Na[superscript +] and K[superscript +] translocation coupled to ATP hydrolysis by the (Na, K)-ATPase, a P[subscript 2c]-type ATPase, or ATP-powered ion pump that actively translocates cations across plasma membranes. The enzyme is also…

Glycemic Variability Is Associated With Reduced Cardiac Autonomic Modulation in Women With Type 2 Diabetes

DEFF Research Database (Denmark)

Fleischer, Jesper; Lebech Cichosz, Simon; Hoeyem, Pernille

2015-01-01

OBJECTIVE: To investigate the sex differences in cardiac autonomic modulation in patients with newly diagnosed type 2 diabetes and to determine whether cardiac autonomic modulation is associated with glycemic variability. RESEARCH DESIGN AND METHODS: We investigated a cohort consisting of 48 men...... and 39 women with non-insulin-treated type 2 diabetes and a known duration of diabetes ... by the standard deviation of normal-to-normal intervals (P = 0.001), the root mean square of successive differences (P = 0.018), LF (P power (P = 0.008), RS ratio (P = 0.027), and expiration-to-inspiration ratio (P = 0.006) was significantly associated with increased glycemic...

Reversible pH-dependent activation/inactivation of CF(1-ATPase of spinach chloroplasts

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A. P. Khomochkin

2017-08-01

Full Text Available The aim of the work was to study the reverse pH-dependent regulation of the enzymatic activity of the catalytic part of ATP synthase (EC 3.6.3.14 of chloroplast – coupling factor CF1. It was shown that the short-term incubation of isolated CF1 in the media with pH 4.5 or 3.5 leads to inactivation of Ca2+-ATPase, which is rapidly (t1/2 ~ 1 min restored in the medium containing 0.5-10 mM bicarbonate at pH 7.8. After acid treatment, the rate of Mg2+-ATPase reaction was also stimulated in the presence of 1 mM bicarbonate (рН 7.8; 37 °С. The increase in Ca2+– and Mg2+-АТР activity of CF1 associated with the addition of NaHCO3 solution was completely eliminated after the introduction of 50 mM acetazolamide – a specific inhibitor of carbonic anhydrase. The obtained results suggest the existence of the bound bicarbonate in the CF1 structure, which apparently participates in proton transfer.

Characteristics of a (Na+K+)-ATPase inhibitor in extracts of tea

International Nuclear Information System (INIS)

Sagnella, G.A.; MacGregor, G.A.

1984-01-01

Extracts of tea were examined for inhibitors of the sodium-potassium pump by investigating the effect of the extracts on 1) isolated preparations of (Na + -K + )-ATPase from hog brain and human blood cells; 2) the displacement of radioactive ouabain from its specific receptor on red blood cells, and 3) the uptake of radioactive rubidium in intact red blood cells. It has been found that extracts of tea were potent inhibitors of the purified hog brain (Na + -K + )-ATPase. However, the inhibition was not specific for the (Na + -K + )-ATPase and the extract of tea did not displace 3 H-ouabain in a specific ouabain-receptor assay. Additionally, the tea extracts displayed only a small inhibitory effect on the uptake of 86 Rb in intact red blood cells. These observations suggest that the material is not like digitalis and that, unlike cardiac glycosides, it may inhibit the activity of the (Na + -K + )-ATPase by interacting with the enzyme at intracellular sites

The gastric H, K-ATPase system also functions as the Na, K-ATPase and Ca-ATPase in altered states [v1; ref status: indexed, http://f1000r.es/1eo

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Tushar Ray

2013-07-01

Full Text Available This article offers an explanation for the apparent lack of Na, K-ATPase activity in parietal cells although ouabain has been known to inhibit gastric acid secretion since 1962. The gastric H, K-ATPase (proton-pump seems to be acting in altered states, thus behaving like a Na, K-ATPase (Na-pump and/or Ca-ATPase (Ca-pump depending on cellular needs.  This conclusion is based on the following findings. First, parietal cell fractions do not exhibit Na, K-ATPase activity at pH 7.0 but do at pH 8.5. Second, the apical plasma membrane (APM fraction exhibits a (Ca or Mg-ATPase activity with negligible H, K-ATPase activity. However, when assayed with Mg alone in presence of the 80 k Da cytosolic proton-pump activator (HAF, the APM fraction reveals remarkably high H, K-ATPase activity, suggesting the observed low affinity of Ca (or Mg-ATPase is an altered state of the latter. Third, calcium (between 1 and 4 µM shows both stimulation and inhibition of the HAF-stimulated H, K-ATPase depending on its concentration, revealing a close interaction between the  proton-pump activator and local Ca concentration in gastric H, K-ATPase function. Such interactions suggest that Ca is acting as a terminal member of the intracellular signaling system for the HAF-regulated proton-pump. It appears that during resting state, the HAF-associated H, K-ATPase remains inhibited by Ca (>1 µM and, prior to resumption of acid secretion the gastric H, K-ATPase acts temporarily as a Ca-pump for removing excess Ca from its immediate environment. This conclusion is consistent with the recent reports of immunochemical co-localization of the gastric H, K-ATPase and Ca-ATPase by superimposition in parietal cells, and a transitory efflux of Ca immediately preceding the onset of acid secretion. These new perspectives on proton-pump function would open new avenues for a fuller understanding of the intracellular regulation of the ubiquitous Na-pump.

The promiscuous phosphomonoestearase activity of Archaeoglobus fulgidus CopA, a thermophilic Cu+ transport ATPase.

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Bredeston, Luis M; González Flecha, F Luis

2016-07-01

Membrane transport P-type ATPases display two characteristic enzymatic activities: a principal ATPase activity provides the driving force for ion transport across biological membranes, whereas a promiscuous secondary activity catalyzes the hydrolysis of phosphate monoesters. This last activity is usually denoted as the phosphatase activity of P-ATPases. In the present study, we characterize the phosphatase activity of the Cu(+)-transport ATPase from Archaeglobus fulgidus (Af-CopA) and compare it with the principal ATPase activity. Our results show that the phosphatase turnover number was 20 times higher than that corresponding to the ATPase activity, but it is compensated by a high value of Km, producing a less efficient catalysis for pNPP. This secondary activity is enhanced by Mg(2+) (essential activator) and phospholipids (non-essential activator), and inhibited by salts and Cu(+). Transition state analysis of the catalyzed and noncatalyzed hydrolysis of pNPP indicates that Af-CopA enhances the reaction rates by a factor of 10(5) (ΔΔG(‡)=38 kJ/mol) mainly by reducing the enthalpy of activation (ΔΔH(‡)=30 kJ/mol), whereas the entropy of activation is less negative on the enzyme than in solution. For the ATPase activity, the decrease in the enthalpic component of the barrier is higher (ΔΔH(‡)=39 kJ/mol) and the entropic component is small on both the enzyme and in solution. These results suggest that different mechanisms are involved in the transference of the phosphoryl group of p-nitrophenyl phosphate and ATP. Copyright © 2016 Elsevier B.V. All rights reserved.

Regulatory effects of phospholamban on cardiac sarcoplasmic reticulum function

International Nuclear Information System (INIS)

Kim, Hae Won.

1989-01-01

In this thesis, the author reports the effect of phospholamban on: (a) Ca 2+ release by cardiac SR and (b) the Ca 2+ -ATPase activity in a purified reconstituted system. Phosphorylation of phospholamban by Ca 2+ · calmodulin-dependent protein kinase had no appreciable effect on the initial rates of Ca 2+ release from cardiac SR vesicles loaded under passive conditions and on the apparent 45 Ca 2+ - 40 Ca 2+ exchange from cardiac SR vesicles loaded under active conditions. us, it appears that Ca 2+ · calmodulin-dependent phosphorylation of phospholamban is not involved in the regulation of Ca 2+ release and 45 Ca 2+-40 Ca 2+ exchange. To determine the molecular mechanism by which phospholamban regulates the Ca 2+ pump, a reconstituted system was developed, using a freeze-thaw sonication procedure. The Ca 2+ -ATPase was purified by a method which yields an active enzyme preparation essentially free of phospholamban. The best rates of Ca 2+ uptake were obtained when cholate and phosphatidylcholine (PC) were used at a ratio of cholate/PC/Ca 2 + -ATPase of 2/80/1. The maximal rates of Ca 2+ Uptake were 700 nmol/min/mg reconstituted vesicles compared to 800 nmol/min/mg SR vesicles. The EC 50 values for Ca 2+ were 0.05 μM for both Ca 2+ uptake and Ca 2+ -ATPase activity in the reconstituted vesicles compared to 0.63 μM Ca 2+ in native SR vesicles. To determine the effect of phospholamban on the Ca + -ATPase activity in the reconstituted vesicles, purified phospholamban was added to the cholate/Ca 2+ -ATPase mixture prior to combining it with liposomes

Crystal structure of a Na+-bound Na+,K+-ATPase preceding the E1P state.

Science.gov (United States)

Kanai, Ryuta; Ogawa, Haruo; Vilsen, Bente; Cornelius, Flemming; Toyoshima, Chikashi

2013-10-10

Na(+),K(+)-ATPase pumps three Na(+) ions out of cells in exchange for two K(+) taken up from the extracellular medium per ATP molecule hydrolysed, thereby establishing Na(+) and K(+) gradients across the membrane in all animal cells. These ion gradients are used in many fundamental processes, notably excitation of nerve cells. Here we describe 2.8 Å-resolution crystal structures of this ATPase from pig kidney with bound Na(+), ADP and aluminium fluoride, a stable phosphate analogue, with and without oligomycin that promotes Na(+) occlusion. These crystal structures represent a transition state preceding the phosphorylated intermediate (E1P) in which three Na(+) ions are occluded. Details of the Na(+)-binding sites show how this ATPase functions as a Na(+)-specific pump, rejecting K(+) and Ca(2+), even though its affinity for Na(+) is low (millimolar dissociation constant). A mechanism for sequential, cooperative Na(+) binding can now be formulated in atomic detail.

Structure and Function of Cu(I)- and Zn(II)-ATPases

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Sitsel, Oleg; Grønberg, Christina; Autzen, Henriette Elisabeth

2015-01-01

Copper and zinc are micronutrients essential for the function of many enzymes while also being toxic at elevated concentrations. Cu(I)- and Zn(II)-transporting P-type ATPases of subclass 1B are of key importance for the homeostasis of these transition metals, allowing ion transport across cellular...... membranes at the expense of ATP. Recent biochemical studies and crystal structures have significantly improved our understanding of the transport mechanisms of these proteins, but many details about their structure and function remain elusive. Here we compare the Cu(I)- and Zn(II)-ATPases, scrutinizing...

Pregestational type 2 diabetes mellitus induces cardiac hypertrophy in the murine embryo through cardiac remodeling and fibrosis.

Science.gov (United States)

Lin, Xue; Yang, Penghua; Reece, E Albert; Yang, Peixin

2017-08-01

activated the proapoptotic c-Jun-N-terminal kinase 1/2 stress signaling and triggered cell apoptosis by increasing the number of terminal deoxynucleotidyl transferase 2'-deoxyuridine 5'-triphosphate nick end labeling-positive cells (10.4 ± 2.2% of the type 2 diabetes mellitus group vs 3.8 ± 0.7% of the nondiabetic group, P type 2 diabetes mellitus induces cardiac hypertrophy in embryonic hearts. Adverse cardiac remodeling, including elevated collagen synthesis, suppressed fibronectin synthesis, profibrosis, and apoptosis, is implicated as the etiology of cardiac hypertrophy. Copyright © 2017 Elsevier Inc. All rights reserved.

Sodium ions as substitutes for protons in the gastric H,K-ATPase

International Nuclear Information System (INIS)

Polvani, C.; Sachs, G.; Blostein, R.

1989-01-01

In view of the striking homology among various ion-translocating ATPases including Na,K-ATPase, Ca-ATPase, and H,K-ATPase, and the recent evidence that protons can replace cytoplasmic sodium as well as potassium in the reaction mechanism of the Na,K-ATPase (Polvani, C., and Blostein, R. (1988) J. Biol. Chem. 263, 16757-16763), we studied the role of sodium as a substitute for protons in the H,K-ATPase reaction. Using hog gastric H,K-ATPase-rich inside-out membrane vesicles we observed 22Na+ influx which was stimulated by intravesicular potassium ions (K+i) at pH 8.5 but not at pH 7.1. This sodium influx was observed in medium containing ATP and was inhibited by vanadate and SCH28080, a selective inhibitor of the gastric H,K-ATPase. At least 2-fold accumulation of sodium was observed at pH 8.5. Experiments aimed to determine the sidedness of the alkaline pH requirement for K+i-dependent sodium influx showed that K+i-activated sodium influx depends on pHout and is unaffected by changes in pHin. These results support the conclusion that sodium ions substitute for protons in the H,K-ATPase reaction mechanism and provide evidence for a similarity in ion selectivity and/or binding domains of the Na,K-ATPase and the gastric H,K-ATPase enzymes

V-ATPase as an effective therapeutic target for sarcomas

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Perut, Francesca, E-mail: francesca.perut@ior.it [Laboratory for Orthopaedic Pathophysiology and Regenerative Medicine, Istituto Ortopedico Rizzoli, Bologna (Italy); Avnet, Sofia; Fotia, Caterina; Baglìo, Serena Rubina; Salerno, Manuela [Laboratory for Orthopaedic Pathophysiology and Regenerative Medicine, Istituto Ortopedico Rizzoli, Bologna (Italy); Hosogi, Shigekuni [Laboratory for Orthopaedic Pathophysiology and Regenerative Medicine, Istituto Ortopedico Rizzoli, Bologna (Italy); Department of Molecular Cell Physiology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto (Japan); Kusuzaki, Katsuyuki [Department of Molecular Cell Physiology, Graduate School of Medical Science, Kyoto Prefectural University of Medicine, Kyoto (Japan); Baldini, Nicola [Laboratory for Orthopaedic Pathophysiology and Regenerative Medicine, Istituto Ortopedico Rizzoli, Bologna (Italy); Department of Biomedical and Neuromotor Sciences, University of Bologna, Bologna (Italy)

2014-01-01

Malignant tumors show intense glycolysis and, as a consequence, high lactate production and proton efflux activity. We investigated proton dynamics in osteosarcoma, rhabdomyosarcoma, and chondrosarcoma, and evaluated the effects of esomeprazole as a therapeutic agent interfering with tumor acidic microenvironment. All sarcomas were able to survive in an acidic microenvironment (up to 5.9–6.0 pH) and abundant acidic lysosomes were found in all sarcoma subtypes. V-ATPase, a proton pump that acidifies intracellular compartments and transports protons across the plasma membrane, was detected in all cell types with a histotype-specific expression pattern. Esomeprazole administration interfered with proton compartmentalization in acidic organelles and induced a significant dose-dependent toxicity. Among the different histotypes, rhabdomyosarcoma, expressing the highest levels of V-ATPase and whose lysosomes are most acidic, was mostly susceptible to ESOM treatment. - Highlights: • Osteosarcoma, rhabdomyosarcoma, and chondrosarcoma survive in acidic microenvironment. • At acidic extracellular pH, sarcoma survival is dependent on V-ATPase expression. • Esomeprazole administration induce a significant dose-dependent toxicity.

The parietal cell gastric H, K-ATPase also functions as the Na, K-ATPase and Ca-ATPase in altered states [v2; ref status: indexed, http://f1000r.es/1tc

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Tushar Ray

2013-09-01

Full Text Available This article offers an explanation for the apparent lack of Na, K-ATPase activity in parietal cells although ouabain has been known to inhibit gastric acid secretion since 1962. The gastric H, K-ATPase (proton-pump seems to be acting in altered states, thus behaving like a Na, K-ATPase (Na-pump and/or Ca-ATPase (Ca-pump depending on cellular needs.  This conclusion is based on the following findings. First, parietal cell fractions do not exhibit Na, K-ATPase activity at pH 7.0 but do at pH 8.5. Second, the apical plasma membrane (APM fraction exhibits a (Ca or Mg-ATPase activity with negligible H, K-ATPase activity. However, when assayed with Mg alone in presence of the 80 k Da cytosolic proton-pump activator (HAF, the APM fraction reveals remarkably high H, K-ATPase activity, suggesting the observed low affinity of Ca (or Mg-ATPase is an altered state of the latter. Third, calcium (between 1 and 4 µM shows both stimulation and inhibition of the HAF-stimulated H, K-ATPase depending on its concentration, revealing a close interaction between the  proton-pump activator and local Ca concentration in gastric H, K-ATPase function. Such interactions suggest that Ca is acting as a terminal member of the intracellular signaling system for the HAF-regulated proton-pump. It appears that during resting state, the HAF-associated H, K-ATPase remains inhibited by Ca (>1 µM and, prior to resumption of acid secretion the gastric H, K-ATPase acts temporarily as a Ca-pump for removing excess Ca from its immediate environment. This conclusion is consistent with the recent reports of immunochemical co-localization of the gastric H, K-ATPase and Ca-ATPase by superimposition in parietal cells, and a transitory efflux of Ca immediately preceding the onset of acid secretion. These new perspectives on proton-pump function would open new avenues for a fuller understanding of the intracellular regulation of the ubiquitous Na-pump.

Cu/Zn superoxide dismutase and the proton ATPase Pma1p of Saccharomyces cerevisiae

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Baron, J. Allen; Chen, Janice S.; Culotta, Valeria C., E-mail: vculott1@jhu.edu

2015-07-03

In eukaryotes, the Cu/Zn containing superoxide dismutase (SOD1) plays a critical role in oxidative stress protection as well as in signaling. We recently demonstrated a function for Saccharomyces cerevisiae Sod1p in signaling through CK1γ casein kinases and identified the essential proton ATPase Pma1p as one likely target. The connection between Sod1p and Pma1p was explored further by testing the impact of sod1Δ mutations on cells expressing mutant alleles of Pma1p that alter activity and/or post-translational regulation of this ATPase. We report here that sod1Δ mutations are lethal when combined with the T912D allele of Pma1p in the C-terminal regulatory domain. This “synthetic lethality” was reversed by intragenic suppressor mutations in Pma1p, including an A906G substitution that lies within the C-terminal regulatory domain and hyper-activates Pma1p. Surprisingly the effect of sod1Δ mutations on Pma1-T912D is not mediated through the Sod1p signaling pathway involving the CK1γ casein kinases. Rather, Sod1p sustains life of cells expressing Pma1-T912D through oxidative stress protection. The synthetic lethality of sod1Δ Pma1-T912D cells is suppressed by growing cells under low oxygen conditions or by treatments with manganese-based antioxidants. We now propose a model in which Sod1p maximizes Pma1p activity in two ways: one involving signaling through CK1γ casein kinases and an independent role for Sod1p in oxidative stress protection. - Highlights: • In yeast, the anti-oxidant enzyme SOD1 promotes activity of the proton ATPase Pma1p. • Cells expressing a T912D variant of Pma1p are not viable without SOD1. • SOD1 is needed to protect Pma1-T912D expressing cells from severe oxidative damage. • SOD1 activates Pma1p through casein kinase signaling and oxidative stress protection.

Cu/Zn superoxide dismutase and the proton ATPase Pma1p of Saccharomyces cerevisiae

International Nuclear Information System (INIS)

Baron, J. Allen; Chen, Janice S.; Culotta, Valeria C.

2015-01-01

In eukaryotes, the Cu/Zn containing superoxide dismutase (SOD1) plays a critical role in oxidative stress protection as well as in signaling. We recently demonstrated a function for Saccharomyces cerevisiae Sod1p in signaling through CK1γ casein kinases and identified the essential proton ATPase Pma1p as one likely target. The connection between Sod1p and Pma1p was explored further by testing the impact of sod1Δ mutations on cells expressing mutant alleles of Pma1p that alter activity and/or post-translational regulation of this ATPase. We report here that sod1Δ mutations are lethal when combined with the T912D allele of Pma1p in the C-terminal regulatory domain. This “synthetic lethality” was reversed by intragenic suppressor mutations in Pma1p, including an A906G substitution that lies within the C-terminal regulatory domain and hyper-activates Pma1p. Surprisingly the effect of sod1Δ mutations on Pma1-T912D is not mediated through the Sod1p signaling pathway involving the CK1γ casein kinases. Rather, Sod1p sustains life of cells expressing Pma1-T912D through oxidative stress protection. The synthetic lethality of sod1Δ Pma1-T912D cells is suppressed by growing cells under low oxygen conditions or by treatments with manganese-based antioxidants. We now propose a model in which Sod1p maximizes Pma1p activity in two ways: one involving signaling through CK1γ casein kinases and an independent role for Sod1p in oxidative stress protection. - Highlights: • In yeast, the anti-oxidant enzyme SOD1 promotes activity of the proton ATPase Pma1p. • Cells expressing a T912D variant of Pma1p are not viable without SOD1. • SOD1 is needed to protect Pma1-T912D expressing cells from severe oxidative damage. • SOD1 activates Pma1p through casein kinase signaling and oxidative stress protection

The Wheat E Subunit of V-Type H+-ATPase Is Involved in the Plant Response to Osmotic Stress

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Xiao-Hong Zhang

2014-09-01

Full Text Available The vacuolar type H+-ATPase (V-type H+-ATPase plays important roles in establishing an electrochemical H+-gradient across tonoplast, energizing Na+ sequestration into the central vacuole, and enhancing salt stress tolerance in plants. In this paper, a putative E subunit of the V-type H+-ATPase gene, W36 was isolated from stress-induced wheat de novo transcriptome sequencing combining with 5'-RACE and RT-PCR methods. The full-length of W36 gene was 1097 bp, which contained a 681 bp open reading frame (ORF and encoded 227 amino acids. Southern blot analysis indicated that W36 was a single-copy gene. The quantitative real-time PCR (qRT-PCR analysis revealed that the expression level of W36 could be upregulated by drought, cold, salt, and exogenous ABA treatment. A subcellular localization assay showed that the W36 protein accumulated in the cytoplasm. Isolation of the W36 promoter revealed some cis-acting elements responding to abiotic stresses. Transgenic Arabidopsis plants overexpressing W36 were enhanced salt and mannitol tolerance. These results indicate that W36 is involved in the plant response to osmotic stress.

Evaluating the Cancer Therapeutic Potential of Cardiac Glycosides

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José Manuel Calderón-Montaño

2014-01-01

Full Text Available Cardiac glycosides, also known as cardiotonic steroids, are a group of natural products that share a steroid-like structure with an unsaturated lactone ring and the ability to induce cardiotonic effects mediated by a selective inhibition of the Na+/K+-ATPase. Cardiac glycosides have been used for many years in the treatment of cardiac congestion and some types of cardiac arrhythmias. Recent data suggest that cardiac glycosides may also be useful in the treatment of cancer. These compounds typically inhibit cancer cell proliferation at nanomolar concentrations, and recent high-throughput screenings of drug libraries have therefore identified cardiac glycosides as potent inhibitors of cancer cell growth. Cardiac glycosides can also block tumor growth in rodent models, which further supports the idea that they have potential for cancer therapy. Evidence also suggests, however, that cardiac glycosides may not inhibit cancer cell proliferation selectively and the potent inhibition of tumor growth induced by cardiac glycosides in mice xenografted with human cancer cells is probably an experimental artifact caused by their ability to selectively kill human cells versus rodent cells. This paper reviews such evidence and discusses experimental approaches that could be used to reveal the cancer therapeutic potential of cardiac glycosides in preclinical studies.

Cadmium, ATPase-P, yeast. From transport to toxicity

International Nuclear Information System (INIS)

Gardarin, Aurelie

2007-01-01

Two projects has been developed during my PhD. One consisting in the functional study of CadA, the Cd 2+ -ATPase from Listeria monocytogenes, the other one was focused on the toxicity of cadmium and the associated response of the yeast Saccharomyces cerevisiae. This two studies used a a phenotype of sensitivity to cadmium induced by CadA expression in yeast. This phenotype was used as a screening tool to identify essential amino acids of Cd transport by CadA and to study cadmium toxicity and the corresponding yeast cellular response. CadA actively transports Cd using ATP hydrolysis as energy source. Directed mutagenesis of the membranous polar, sulphur and charged amino-acids revealed that Cd transport pathway implied four transmembrane segments (Tm) and more precisely the cysteine C 354 , C 356 and proline P 355 of the CPC motif located in Tm6, aspartate D 692 in Tm8, glutamate E 164 in Tm4 and methionine M 149 in Tm5. From our studies, 2 Cd ions would be translocated for each hydrolysis ATP. Expression of CadA in the yeast Saccharomyces cerevisiae induces an hypersensitivity to Cd. A wild type cell can grow up to 100 μm cadmium whereas CadA expressing yeast cannot grow with 1 μm cadmium in the culture medium. This cadmium sensitivity was due to the localisation of CadA in the endoplasmic reticulum membrane. Transport of cadmium in this compartment produces an accumulation of mis-folded proteins that induces the Unfolded Protein Response (UPR). As UPR also occurs in a wild type yeast exposed to low Cd concentration, one can point out endoplasmic reticulum as a extremely sensitive cellular compartment. UPR also appears as an early response to Cd as it happens far before any visible signs of toxicity. (author) [fr

Rhodamine Inhibitors of P-glycoprotein: An Amide/Thioamide “Switch” for ATPase Activity

Science.gov (United States)

Gannon, Michael K.; Holt, Jason J.; Bennett, Stephanie M.; Wetzel, Bryan R.; Loo, Tip W.; Bartlett, M. Claire; Clarke, David M.; Sawada, Geri A.; Higgins, J. William; Tombline, Gregory; Raub, Thomas J.; Detty, Michael R.

2012-01-01

We have examined 46 tetramethylrosamine/rhodamine derivatives with structural diversity in the heteroatom of the xanthylium core, the amino substituents of the 3- and 6-positions, and the alkyl, aryl, or heteroaryl group at the 9-substituent. These compounds were examined for affinity and ATPase stimulation in isolated MDR3 CL P-gp and human P-gp-His10, for their ability to promote uptake of calcein AM and vinblastine in multidrug-resistant MDCKII-MDR1 cells, and for transport in monolayers of MDCKII-MDR1 cells. Thioamide 31-S gave KM of 0.087 μM in human P-gp. Small changes in structure among this set of compounds affected affinity as well as transport rate (or flux) even though all derivatives examined were substrates for P-gp. With isolated protein, tertiary amide groups dictate high affinity and high stimulation while tertiary thioamide groups give high affinity and inhibition of ATPase activity. In MDCKII-MDR1 cells, the tertiary thioamide-containing derivatives promote uptake of calcein AM and have very slow passive, absorptive, and secretory rates of transport relative to transport rates for tertiary amide-containing derivatives. Thioamide 31-S promoted uptake of calcein AM and inhibited efflux of vinblastine with IC50’s of ~2 μM in MDCKII-MDR1 cells. PMID:19402665

Na+/K+-ATPase is present in scrapie-associated fibrils, modulates PrP misfolding in vitro and links PrP function and dysfunction.

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James F Graham

Full Text Available Transmissible spongiform encephalopathies are characterised by widespread deposition of fibrillar and/or plaque-like forms of the prion protein. These aggregated forms are produced by misfolding of the normal prion protein, PrP(C, to the disease-associated form, PrP(Sc, through mechanisms that remain elusive but which require either direct or indirect interaction between PrP(C and PrP(Sc isoforms. A wealth of evidence implicates other non-PrP molecules as active participants in the misfolding process, to catalyse and direct the conformational conversion of PrP(C or to provide a scaffold ensuring correct alignment of PrP(C and PrP(Sc during conversion. Such molecules may be specific to different scrapie strains to facilitate differential prion protein misfolding. Since molecular cofactors may become integrated into the growing protein fibril during prion conversion, we have investigated the proteins contained in prion disease-specific deposits by shotgun proteomics of scrapie-associated fibrils (SAF from mice infected with 3 different strains of mouse-passaged scrapie. Concomitant use of negative control preparations allowed us to identify and discount proteins that are enriched non-specifically by the SAF isolation protocol. We found several proteins that co-purified specifically with SAF from infected brains but none of these were reproducibly and demonstrably specific for particular scrapie strains. The α-chain of Na(+/K(+-ATPase was common to SAF from all 3 strains and we tested the ability of this protein to modulate in vitro misfolding of recombinant PrP. Na(+/K(+-ATPase enhanced the efficiency of disease-specific conversion of recombinant PrP suggesting that it may act as a molecular cofactor. Consistent with previous results, the same protein inhibited fibrillisation kinetics of recombinant PrP. Since functional interactions between PrP(C and Na(+/K(+-ATPase have previously been reported in astrocytes, our data highlight this molecule as

Cardiac function improved by sarcoplasmic reticulum Ca2+-ATPase overexpression in a heart failure model induced by chronic myocardial ischemia

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Wei XIN

2011-04-01

Full Text Available Objective Chronic myocardial ischemia(CMI has become an important cause of heart failure(HF.The aim of present study was to examine the effects of Sarco-endoplasmic reticulum calcium ATPase(SERCA2a gene transfer in HF model in large animal induced by CMI.Methods HF was reproduced in minipigs by ligating the initial segment of proximal left anterior descending(LAD coronary artery with an ameroid constrictor to produce progressive vessel occlusion and ischemia.After confirmation of myocardial perfusion defect and cardiac function impairment by SPECT and echocardiography in the model,animals were divided into 4 groups: HF group;HF+enhanced green fluorescent protein(EGFP group;HF+SERCA2a group;and sham operation group as control.rAAV1-EGFP and rAAV1-SERCA2a(1×1012 vg for each animal were directly and intramyocardially injected to the animals of HF+EGFP and HF+SERCA2a groups.Sixty days after the gene transfer,the expression of SERCA2a at the protein level was examined by Western blotting and immunohistochemistry,the changes in cardiac function were determined by echocardiographic and hemodynamic analysis,and the changes in serum inflammatory and neuro-hormonal factors(including BNP,TNF-a,IL-6,ET-1 and Ang II were determined by radioimmunoassay.Results Sixty days after gene transfer,LVEF,Ev/Av and ±dp/dtmax increased significantly(P < 0.05,along with an increase of SERCA2a protein expression in the ischemic myocardium(PP < 0.05,accompanied by a significant decrease of inflammatory and neural-hormonal factors(PP < 0.05 in HF+SERCA2a group as compared with HF/HF+EGFP group.Conclusions Overexpression of SERCA2a may significantly improve the cardiac function of the ischemic myocardium of HF model induced by CMI and reverse the activation of neural-hormonal factors,implying that it has a potential therapeutic significance in CMI related heart failure.

P-glycoprotein ATPase activity requires lipids to activate a switch at the first transmission interface.

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Loo, Tip W; Clarke, David M

2016-04-01

P-glycoprotein (P-gp) is an ABC (ATP-Binding Cassette) drug pump. A common feature of ABC proteins is that they are organized into two wings. Each wing contains a transmembrane domain (TMD) and a nucleotide-binding domain (NBD). Drug substrates and ATP bind at the interface between the TMDs and NBDs, respectively. Drug transport involves ATP-dependent conformational changes between inward- (open, NBDs far apart) and outward-facing (closed, NBDs close together) conformations. P-gps crystallized in the presence of detergent show an open structure. Human P-gp is inactive in detergent but basal ATPase activity is restored upon addition of lipids. The lipids might cause closure of the wings to bring the NBDs close together to allow ATP hydrolysis. We show however, that cross-linking the wings together did not activate ATPase activity when lipids were absent suggesting that lipids may induce other structural changes required for ATPase activity. We then tested the effect of lipids on disulfide cross-linking of mutants at the first transmission interface between intracellular loop 4 (TMD2) and NBD1. Mutants L443C/S909C and L443C/R905C but not G471C/S909C and V472C/S909C were cross-linked with oxidant when in membranes. The mutants were then purified and cross-linked with or without lipids. Mutants G471C/S909C and V472C/S909C cross-linked only in the absence of lipids whereas mutants L443C/S909C and L443C/R905C were cross-linked only in the presence of lipids. The results suggest that lipids activate a switch at the first transmission interface and that the structure of P-gp is different in detergents and lipids. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

Novel protective role of endogenous cardiac myocyte P2X4 receptors in heart failure.

Science.gov (United States)

Yang, Tiehong; Shen, Jian-bing; Yang, Ronghua; Redden, John; Dodge-Kafka, Kimberly; Grady, James; Jacobson, Kenneth A; Liang, Bruce T

2014-05-01

Heart failure (HF), despite continuing progress, remains a leading cause of mortality and morbidity. P2X4 receptors (P2X4R) have emerged as potentially important molecules in regulating cardiac function and as potential targets for HF therapy. Transgenic P2X4R overexpression can protect against HF, but this does not explain the role of native cardiac P2X4R. Our goal is to define the physiological role of endogenous cardiac myocyte P2X4R under basal conditions and during HF induced by myocardial infarction or pressure overload. Mice established with conditional cardiac-specific P2X4R knockout were subjected to left anterior descending coronary artery ligation-induced postinfarct or transverse aorta constriction-induced pressure overload HF. Knockout cardiac myocytes did not show P2X4R by immunoblotting or by any response to the P2X4R-specific allosteric enhancer ivermectin. Knockout hearts showed normal basal cardiac function but depressed contractile performance in postinfarct and pressure overload models of HF by in vivo echocardiography and ex vivo isolated working heart parameters. P2X4R coimmunoprecipitated and colocalized with nitric oxide synthase 3 (eNOS) in wild-type cardiac myocytes. Mice with cardiac-specific P2X4R overexpression had increased S-nitrosylation, cyclic GMP, NO formation, and were protected from postinfarct and pressure overload HF. Inhibitor of eNOS, L-N(5)-(1-iminoethyl)ornithine hydrochloride, blocked the salutary effect of cardiac P2X4R overexpression in postinfarct and pressure overload HF as did eNOS knockout. This study establishes a new protective role for endogenous cardiac myocyte P2X4R in HF and is the first to demonstrate a physical interaction between the myocyte receptor and eNOS, a mediator of HF protection. © 2014 American Heart Association, Inc.

Impact of Gastric H+/K+-ATPase rs2733743 on the Intragastric pH-Values of Dexlansoprazole Injection in Chinese Subjects

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Lu-Ning Sun

2017-09-01

Full Text Available Background: Not all patients with acid-related disorders receiving proton pump inhibitor (PP treatment get adequate gastric pH control. The genetic variation of receptors, metabolic enzymes, and transporters are known to cause failures of therapies. We have conducted a study to evaluate the influence of gastric H+/K+-ATPase, CYP2C19, and ABCB1 polymorphisms on the pharmacokinetic and pharmacodynamic profiles of dexlansoprazole injection in healthy Chinese subjects.Methods: A total of 51 subjects were enrolled for pharmacokinetic and pharmacodynamic study after a single intravenous administration of 20 or 30 mg dexlansoprazole. Plasma concentrations were determined using a chiral liquid chromatography-mass spectrometry method. The intragastric pH and baseline-adjusted intragastric pH parameters were introduced to evaluate the pharmacodynamic characters. Genotyping was performed by polymerase chain reaction.Results: The pharmacokinetic parameters were significantly influenced by CYP2C19 phenotypes, and gastric acid secretion inhibition were affected by both gastric H+/K+-ATPase and CYP2C19 polymorphisms. Gastric H+/K+-ATPase genotypes had greater effects than CYP2C19 genotypes on the suppression of gastric acid secretion.Conclusion: Gastric H+/K+-ATPase polymorphism may be one of the main reasons that cause insufficient gastric acid inhibition.

Epicardial, pericardial and total cardiac fat and cardiovascular disease in type 2 diabetic patients with elevated urinary albumin excretion rate

DEFF Research Database (Denmark)

Christensen, Regitse H.; Von Scholten, Bernt J.; Hansen, Christian S.

2017-01-01

of 200 patients with type 2 diabetes and elevated urinary albumin excretion rate (UAER). Methods Cardiac adipose tissue was measured from baseline echocardiography. The composite endpoint comprised incident cardiovascular disease and all-cause mortality. Coronary artery calcium, carotid intima media.......7, p = 0.017) models. Cardiac adipose tissue (p = 0.033) was associated with baseline coronary artery calcium (model 1) and interleukin-8 (models 1-3, all p type 2 diabetes patients without coronary artery disease, high cardiac adipose tissue levels were associated...

Crystal structure of the sodium-potassium pump (Na+,K+-ATPase) with bound potassium and ouabain

Science.gov (United States)

Ogawa, Haruo; Shinoda, Takehiro; Cornelius, Flemming; Toyoshima, Chikashi

2009-01-01

The sodium-potassium pump (Na+,K+-ATPase) is responsible for establishing Na+ and K+ concentration gradients across the plasma membrane and therefore plays an essential role in, for instance, generating action potentials. Cardiac glycosides, prescribed for congestive heart failure for more than 2 centuries, are efficient inhibitors of this ATPase. Here we describe a crystal structure of Na+,K+-ATPase with bound ouabain, a representative cardiac glycoside, at 2.8 Å resolution in a state analogous to E2·2K+·Pi. Ouabain is deeply inserted into the transmembrane domain with the lactone ring very close to the bound K+, in marked contrast to previous models. Due to antagonism between ouabain and K+, the structure represents a low-affinity ouabain-bound state. Yet, most of the mutagenesis data obtained with the high-affinity state are readily explained by the present crystal structure, indicating that the binding site for ouabain is essentially the same. According to a homology model for the high affinity state, it is a closure of the binding cavity that confers a high affinity. PMID:19666591

Crystal structure of the sodium-potassium pump (Na+,K+-ATPase) with bound potassium and ouabain.

Science.gov (United States)

Ogawa, Haruo; Shinoda, Takehiro; Cornelius, Flemming; Toyoshima, Chikashi

2009-08-18

The sodium-potassium pump (Na(+),K(+)-ATPase) is responsible for establishing Na(+) and K(+) concentration gradients across the plasma membrane and therefore plays an essential role in, for instance, generating action potentials. Cardiac glycosides, prescribed for congestive heart failure for more than 2 centuries, are efficient inhibitors of this ATPase. Here we describe a crystal structure of Na(+),K(+)-ATPase with bound ouabain, a representative cardiac glycoside, at 2.8 A resolution in a state analogous to E2.2K(+).Pi. Ouabain is deeply inserted into the transmembrane domain with the lactone ring very close to the bound K(+), in marked contrast to previous models. Due to antagonism between ouabain and K(+), the structure represents a low-affinity ouabain-bound state. Yet, most of the mutagenesis data obtained with the high-affinity state are readily explained by the present crystal structure, indicating that the binding site for ouabain is essentially the same. According to a homology model for the high affinity state, it is a closure of the binding cavity that confers a high affinity.

Cardiac-specific inducible overexpression of human plasma membrane Ca2+ ATPase 4b is cardioprotective and improves survival in mice following ischemic injury.

Science.gov (United States)

Sadi, Al Muktafi; Afroze, Talat; Siraj, M Ahsan; Momen, Abdul; White-Dzuro, Colin; Zarrin-Khat, Dorrin; Handa, Shivalika; Ban, Kiwon; Kabir, M Golam; Trivieri, Maria G; Gros, Robert; Backx, Peter; Husain, Mansoor

2018-03-30

Background: Heart failure (HF) is associated with reduced expression of plasma membrane Ca 2+ -ATPase 4 (PMCA4). Cardiac-specific overexpression of human PMCA4b in mice inhibited nNOS activity and reduced cardiac hypertrophy by inhibiting calcineurin. Here we examine temporally regulated cardiac-specific overexpression of hPMCA4b in mouse models of myocardial ischemia reperfusion injury (IRI) ex vivo , and HF following experimental myocardial infarction (MI) in vivo Methods and results: Doxycycline-regulated cardiomyocyte-specific overexpression and activity of hPMCA4b produced adaptive changes in expression levels of Ca 2+ -regulatory genes, and induced hypertrophy without significant differences in Ca 2+ transients or diastolic Ca 2+ concentrations. Total cardiac NOS and nNOS-specific activities were reduced in mice with cardiac overexpression of hPMCA4b while nNOS, eNOS and iNOS protein levels did not differ. hMPCA4b-overexpressing mice also exhibited elevated systolic blood pressure vs. controls, with increased contractility and lusitropy in vivo In isolated hearts undergoing IRI, hPMCA4b overexpression was cardioprotective. NO donor-treated hearts overexpressing hPMCA4b showed reduced LVDP and larger infarct size versus vehicle-treated hearts undergoing IRI, demonstrating that the cardioprotective benefits of hPMCA4b-repressed nNOS are lost by restoring NO availability. Finally, both pre-existing and post-MI induction of hPMCA4b overexpression reduced infarct expansion and improved survival from HF. Conclusions: Cardiac PMCA4b regulates nNOS activity, cardiac mass and contractility, such that PMCA4b overexpression preserves cardiac function following IRI, heightens cardiac performance and limits infarct progression, cardiac hypertrophy and HF, even when induced late post-MI. These data identify PMCA4b as a novel therapeutic target for IRI and HF. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

Regulation of vacuolar H{sup +}-ATPase in microglia by RANKL

Energy Technology Data Exchange (ETDEWEB)

Serrano, Eric M.; Ricofort, Ryan D.; Zuo, Jian [Department of Orthodontics, University of Florida College of Dentistry, Gainesville, FL 32610 (United States); Ochotny, Noelle [Department of Pharmacology, University of Toronto, Toronto, Ont., Canada M5G 1G6 (Canada); Manolson, Morris F. [Faculty of Dentistry, University of Toronto, Toronto, Ont., Canada M5G 1G6 (Canada); Holliday, L. Shannon, E-mail: sholliday@dental.ufl.edu [Department of Orthodontics, University of Florida College of Dentistry, Gainesville, FL 32610 (United States); Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, FL 32610 (United States)

2009-11-06

Vacuolar H{sup +}-ATPases (V-ATPases) are large electrogenic proton pumps composed of numerous subunits that play vital housekeeping roles in the acidification of compartments of the endocytic pathway. Additionally, V-ATPases play specialized roles in certain cell types, a capacity that is linked to cell type selective expression of isoforms of some of the subunits. We detected low levels of the a3 isoform of the a-subunit in mouse brain extracts. Examination of various brain-derived cell types by immunoblotting showed a3 was expressed in the N9 microglia cell line and in primary microglia, but not in other cell types. The expression of a3 in osteoclasts requires stimulation by Receptor Activator of Nuclear Factor {kappa}B-ligand (RANKL). We found that Receptor Activator of Nuclear Factor {kappa}B (RANK) was expressed by microglia. Stimulation of microglia with RANKL triggered increased expression of a3. V-ATPases in microglia were shown to bind microfilaments, and stimulation with RANKL increased the proportion of V-ATPase associated with the detergent-insoluble cytoskeletal fraction and with actin. In summary, microglia express the a3-subunit of V-ATPase. The expression of a3 and the interaction between V-ATPases and microfilaments was modulated by RANKL. These data suggest a novel molecular pathway for regulating microglia.

A phytochemical study of the Cuphea glutinosa from Southern Brazil: Na+,K+-ATPase activity inhibition and antioxidant properties.

Science.gov (United States)

Zago, Adriana M; Carvalho, Fabiano B; Gutierres, Jessié Martins; Bohnert, Crystiani; Fernandes, Marilda da Cruz; Morandini, Liziane M; Coelho, Helena S; Fogaça, Aline O; Andrade, Cinthia M; Mostardeiro, Marco A; Dalcol, Ionara I; Morel, Ademir F

2018-05-21

This study investigated the antioxidant activity of Cuphea glutinosa (CG) and its effect on Na + , K + -ATPase from cardiac muscle. The ethanolic extract showed higher antioxidant capacity compared to aqueous and ethyl acetate fraction. Ethyl acetate fraction showed β-sitosterol-3-O-β-glucoside, kaempferol, quercetin, isoquercetin, gallic acid methyl ester, and gallic acid. The ethanolic extract also reduced the Na + ,K + -ATPase activity. CG presented a promising antioxidant activity and inhibitory effect on the Na + , K + -ATPase activity, supporting biochemical evidences the popular use of this plant in the treatment of heart failure.

Spa47 is an oligomerization-activated type three secretion system (T3SS) ATPase from Shigella flexneri.

Science.gov (United States)

Burgess, Jamie L; Jones, Heather B; Kumar, Prashant; Toth, Ronald T; Middaugh, C Russell; Antony, Edwin; Dickenson, Nicholas E

2016-05-01

Gram-negative pathogens often use conserved type three secretion systems (T3SS) for virulence. The Shigella type three secretion apparatus (T3SA) penetrates the host cell membrane and provides a unidirectional conduit for injection of effectors into host cells. The protein Spa47 localizes to the base of the apparatus and is speculated to be an ATPase that provides the energy for T3SA formation and secretion. Here, we developed an expression and purification protocol, producing active Spa47 and providing the first direct evidence that Spa47 is a bona fide ATPase. Additionally, size exclusion chromatography and analytical ultracentrifugation identified multiple oligomeric species of Spa47 with the largest greater than 8 fold more active for ATP hydrolysis than the monomer. An ATPase inactive Spa47 point mutant was then engineered by targeting a conserved Lysine within the predicted Walker A motif of Spa47. Interestingly, the mutant maintained a similar oligomerization pattern as active Spa47, but was unable to restore invasion phenotype when used to complement a spa47 null S. flexneri strain. Together, these results identify Spa47 as a Shigella T3SS ATPase and suggest that its activity is linked to oligomerization, perhaps as a regulatory mechanism as seen in some related pathogens. Additionally, Spa47 catalyzed ATP hydrolysis appears to be essential for host cell invasion, providing a strong platform for additional studies dissecting its role in virulence and providing an attractive target for anti-infective agents. © 2016 The Protein Society.

Analysis of Amino Acid Residues of Potential Importance for Phosphati-dylserine Specificity of P4-type ATPase ATP8A2

DEFF Research Database (Denmark)

Mogensen, Louise; Vestergaard, Anna Lindeløv; Mikkelsen, Stine

The asymmetric structure of the plasma membrane is maintained through internalization of phos-pholipids by the family of P4-ATPases by a poorly characterized mechanism. Studies in yeast point towards a non-classical pathway involving important residues of a two-gate mechanism [1]. Glycine-230...... 302 of ATP8A2 with alanine (N302A), tyrosine (N302Y) and serine (N302S). Furthermore, a triple mutant of ATP8A2 (Q95GQ96AN302S) was studied to reveal any cooperativity between the two gates, as observed in yeast [1]. The affinities of the mutants for phosphatidylserine and phosphatidylethanolamine...

LOCALIZATION OF Na+, K+-ATPASE AND OTHER ENZYMES IN TELEOST PSEUDOBRANCH

Science.gov (United States)

Dendy, Leslie A.; Deter, Russell L.; Philpott, Charles W.

1973-01-01

In an effort to determine the subcellular localization of sodium- and potassium-activated adenosine triphosphatase (Na+, K+-ATPase) in the pseudobranch of the pinfish Lagodon rhomboides, this tissue was fractionated by differential centrifugation and the activities of several marker enzymes in the fractions were measured. Cytochrome c oxidase was found primarily in the mitochondrial-light mitochondrial (M+L) fraction. Phosphoglucomutase appeared almost exclusively in the soluble (S) fraction. Monoamine oxidase was concentrated in the nuclear (N) fraction, with a significant amount also in the microsomal (P) fraction but little in M+L or S. Na+, K+-ATPase and ouabain insensitive Mg2+-ATPase were distributed in N, M+L, and P, the former having its highest specific activity in P and the latter in M+L. Rate sedimentation analysis of the M+L fraction indicated that cytochrome c oxidase and Mg2+-ATPase were associated with a rapidly sedimenting particle population (presumably mitochondria), while Na+, K+-ATPase was found primarily in a slowly sedimenting component. At least 75% of the Na+, K+-ATPase in M+L appeared to be associated with structures containing no Mg2+-ATPase. Kinetic properties of the two ATPases were studied in the P fraction and were typical of these enzymes in other tissues. Na+, K+-ATPase activity was highly dependent on the ratio of Na+ and K+ concentrations but independent of absolute concentrations over at least a fourfold range. PMID:4349221

Reactivity of 12-tungstophosphoric acid and its inhibitor potency toward Na+/K+-ATPase: A combined 31P NMR study, ab initio calculations and crystallographic analysis.

Science.gov (United States)

Bošnjaković-Pavlović, Nada; Bajuk-Bogdanović, Danica; Zakrzewska, Joanna; Yan, Zeyin; Holclajtner-Antunović, Ivanka; Gillet, Jean-Michel; Spasojević-de Biré, Anne

2017-11-01

Influence of 12-tungstophosphoric acid (WPA) on conversion of adenosine triphosphate (ATP) to adenosine diphosphate (ADP) in the presence of Na + /K + -ATPase was monitored by 31 P NMR spectroscopy. It was shown that WPA exhibits inhibitory effect on Na + /K + -ATPase activity. In order to study WPA reactivity and intermolecular interactions between WPA oxygen atoms and different proton donor types (D=O, N, C), we have considered data for WPA based compounds from the Cambridge Structural Database (CSD), the Crystallographic Open Database (COD) and the Inorganic Crystal Structure Database (ICSD). Binding properties of Keggin's anion in biological systems are illustrated using Protein Data Bank (PDB). This work constitutes the first determination of theoretical Bader charges on polyoxotungstate compound via the Atom In Molecule theory. An analysis of electrostatic potential maps at the molecular surface and charge of WPA, resulting from DFT calculations, suggests that the preferred protonation site corresponds to WPA bridging oxygen. These results enlightened WPA chemical reactivity and its potential biological applications such as the inhibition of the ATPase activity. Copyright © 2017 Elsevier Inc. All rights reserved.

Curcumin modulation of Na,K-ATPase: phosphoenzyme accumulation, decreased K+ occlusion, and inhibition of hydrolytic activity

DEFF Research Database (Denmark)

Mahmmoud, Yasser Ahmed

2005-01-01

Curcumin, the major constitute of tumeric, is an important nutraceutical that has been shown to be useful in the treatment of many diseases. As an inhibitor of the sarcoplasmic reticulum Ca2+-ATPase, curcumin was shown to correct cystic fibrosis (CF) defects in some model systems, whereas others...... have reported no or little effects on CF after curcumin treatment, suggesting that curcumin effect is not due to simple inhibition of the Ca2+-ATPase. We tested the hypothesis that curcumin may modulate other members of the P2-type ATPase superfamily by studying the effects of curcumin on the activity...... and kinetic properties of the Na,K-ATPase. Curcumin treatment inhibited Na,K-ATPase activity in a dose-dependent manner (K0.514.6 M). Curcumin decreased the apparent affinity of Na,K-ATPase for K+ and increased it for Na+ and ATP. Kinetic analyses indicated that curcumin induces a three-fold reduction...

Stabilisation of Na,K-ATPase structure by the cardiotonic steroid ouabain

International Nuclear Information System (INIS)

Miles, Andrew J.; Fedosova, Natalya U.; Hoffmann, Søren V.; Wallace, B.A.; Esmann, Mikael

2013-01-01

Highlights: •Ouabain binding to pig and shark Na,K-ATPase enhances thermal stability. •Ouabain stabilises both membrane-bound and solubilised Na,K-ATPase. •Synchrotron radiation circular dichroism is used for structure determination. •Secondary structure in general is not affected by ouabain binding. •Stabilisation is due to re-arrangement of tertiary structure. -- Abstract: Cardiotonic steroids such as ouabain bind with high affinity to the membrane-bound cation-transporting P-type Na,K-ATPase, leading to complete inhibition of the enzyme. Using synchrotron radiation circular dichroism spectroscopy we show that the enzyme-ouabain complex is less susceptible to thermal denaturation (unfolding) than the ouabain-free enzyme, and this protection is observed with Na,K-ATPase purified from pig kidney as well as from shark rectal glands. It is also shown that detergent-solubilised preparations of Na,K-ATPase are stabilised by ouabain, which could account for the successful crystallisation of Na,K-ATPase in the ouabain-bound form. The secondary structure is not significantly affected by the binding of ouabain. Ouabain appears however, to induce a reorganization of the tertiary structure towards a more compact protein structure which is less prone to unfolding; recent crystal structures of the two enzymes are consistent with this interpretation. These circular dichroism spectroscopic studies in solution therefore provide complementary information to that provided by crystallography

Stabilisation of Na,K-ATPase structure by the cardiotonic steroid ouabain

Energy Technology Data Exchange (ETDEWEB)

Miles, Andrew J. [Institute of Structural and Molecular Biology, Birkbeck College, University of London, London WC1E 7HX (United Kingdom); Fedosova, Natalya U. [Department of Biomedicine, Aarhus University, DK-8000 Aarhus (Denmark); Hoffmann, Søren V. [ISA, Department of Physics and Astronomy, Aarhus University, DK-8000 Aarhus (Denmark); Wallace, B.A. [Institute of Structural and Molecular Biology, Birkbeck College, University of London, London WC1E 7HX (United Kingdom); Esmann, Mikael, E-mail: me@biophys.au.dk [Department of Biomedicine, Aarhus University, DK-8000 Aarhus (Denmark)

2013-05-31

Highlights: •Ouabain binding to pig and shark Na,K-ATPase enhances thermal stability. •Ouabain stabilises both membrane-bound and solubilised Na,K-ATPase. •Synchrotron radiation circular dichroism is used for structure determination. •Secondary structure in general is not affected by ouabain binding. •Stabilisation is due to re-arrangement of tertiary structure. -- Abstract: Cardiotonic steroids such as ouabain bind with high affinity to the membrane-bound cation-transporting P-type Na,K-ATPase, leading to complete inhibition of the enzyme. Using synchrotron radiation circular dichroism spectroscopy we show that the enzyme-ouabain complex is less susceptible to thermal denaturation (unfolding) than the ouabain-free enzyme, and this protection is observed with Na,K-ATPase purified from pig kidney as well as from shark rectal glands. It is also shown that detergent-solubilised preparations of Na,K-ATPase are stabilised by ouabain, which could account for the successful crystallisation of Na,K-ATPase in the ouabain-bound form. The secondary structure is not significantly affected by the binding of ouabain. Ouabain appears however, to induce a reorganization of the tertiary structure towards a more compact protein structure which is less prone to unfolding; recent crystal structures of the two enzymes are consistent with this interpretation. These circular dichroism spectroscopic studies in solution therefore provide complementary information to that provided by crystallography.

O2 free radicals: cause of ischemia-reperfusion injury to cardiac Na+-K+-ATPase

International Nuclear Information System (INIS)

Kim, M.S.; Akera, T.

1987-01-01

The role of O2 free radicals in the reduction of sarcolemmal Na+-K+-ATPase, which occurs during reperfusion of ischemic heart, was examined in isolated guinea pig heart using exogenous scavengers of O2 radicals and an inhibitor of xanthine oxidase. Ischemia and reperfusion reduced Na+-K+-ATPase activity and specific [3H]ouabain binding to the enzyme in ventricular muscle homogenates and also markedly lowered sodium pump activity estimated from ouabain-sensitive 86Rb+ uptake by ventricular muscle slices. These effects of ischemia and reperfusion were prevented to various degrees by O2-radical scavengers, such as superoxide dismutase, catalase, dimethyl-sulfoxide, histidine, or vitamin E or by the xanthine oxidase inhibitor, allopurinol. The degree of protection afforded by these agents paralleled that of reduction in enhanced lipid peroxidation of myocardial tissue as estimated from malondialdehyde production. These results strongly suggest that O2 radicals play a crucial role in the injury to sarcolemmal Na+-K+-ATPase during reperfusion of ischemic heart

O2 free radicals: cause of ischemia-reperfusion injury to cardiac Na+-K+-ATPase

Energy Technology Data Exchange (ETDEWEB)

Kim, M.S.; Akera, T.

1987-02-01

The role of O2 free radicals in the reduction of sarcolemmal Na+-K+-ATPase, which occurs during reperfusion of ischemic heart, was examined in isolated guinea pig heart using exogenous scavengers of O2 radicals and an inhibitor of xanthine oxidase. Ischemia and reperfusion reduced Na+-K+-ATPase activity and specific (3H)ouabain binding to the enzyme in ventricular muscle homogenates and also markedly lowered sodium pump activity estimated from ouabain-sensitive 86Rb+ uptake by ventricular muscle slices. These effects of ischemia and reperfusion were prevented to various degrees by O2-radical scavengers, such as superoxide dismutase, catalase, dimethyl-sulfoxide, histidine, or vitamin E or by the xanthine oxidase inhibitor, allopurinol. The degree of protection afforded by these agents paralleled that of reduction in enhanced lipid peroxidation of myocardial tissue as estimated from malondialdehyde production. These results strongly suggest that O2 radicals play a crucial role in the injury to sarcolemmal Na+-K+-ATPase during reperfusion of ischemic heart.

Structural studies of P-type ATPase–ligand complexes using an X-ray free-electron laser

Energy Technology Data Exchange (ETDEWEB)

Bublitz, Maike; Nass, Karol; Drachmann, Nikolaj D.; Markvardsen, Anders J.; Gutmann, Matthias J.; Barends, Thomas R. M.; Mattle, Daniel; Shoeman, Robert L.; Doak, R. Bruce; Boutet, Sébastien; Messerschmidt, Marc; Seibert, Marvin M.; Williams, Garth J.; Foucar, Lutz; Reinhard, Linda; Sitsel, Oleg; Gregersen, Jonas L.; Clausen, Johannes D.; Boesen, Thomas; Gotfryd, Kamil; Wang, Kai-Tuo; Olesen, Claus; Møller, Jesper V.; Nissen, Poul; Schlichting, Ilme

2015-06-11

<p>Membrane proteins are key players in biological systems, mediating signalling events and the specific transport ofe.g.ions and metabolites. Consequently, membrane proteins are targeted by a large number of currently approved drugs. Understanding their functions and molecular mechanisms is greatly dependent on structural information, not least on complexes with functionally or medically important ligands. Structure determination, however, is hampered by the difficulty of obtaining well diffracting, macroscopic crystals. Here, the feasibility of X-ray free-electron-laser-based serial femtosecond crystallography (SFX) for the structure determination of membrane protein–ligand complexes using microcrystals of various native-source and recombinant P-type ATPase complexes is demonstrated. The data reveal the binding sites of a variety of ligands, including lipids and inhibitors such as the hallmark P-type ATPase inhibitor orthovanadate. By analyzing the resolution dependence of ligand densities and overall model qualities, SFX data quality metrics as well as suitable refinement procedures are discussed. Even at relatively low resolution and multiplicity, the identification of ligands can be demonstrated. This makes SFX a useful tool for ligand screening and thus for unravelling the molecular mechanisms of biologically active proteins.p>

Stabilisation of Na,K-ATPase structure by the cardiotonic steroid ouabain.

Science.gov (United States)

Miles, Andrew J; Fedosova, Natalya U; Hoffmann, Søren V; Wallace, B A; Esmann, Mikael

2013-05-31

Cardiotonic steroids such as ouabain bind with high affinity to the membrane-bound cation-transporting P-type Na,K-ATPase, leading to complete inhibition of the enzyme. Using synchrotron radiation circular dichroism spectroscopy we show that the enzyme-ouabain complex is less susceptible to thermal denaturation (unfolding) than the ouabain-free enzyme, and this protection is observed with Na,K-ATPase purified from pig kidney as well as from shark rectal glands. It is also shown that detergent-solubilised preparations of Na,K-ATPase are stabilised by ouabain, which could account for the successful crystallisation of Na,K-ATPase in the ouabain-bound form. The secondary structure is not significantly affected by the binding of ouabain. Ouabain appears however, to induce a reorganization of the tertiary structure towards a more compact protein structure which is less prone to unfolding; recent crystal structures of the two enzymes are consistent with this interpretation. These circular dichroism spectroscopic studies in solution therefore provide complementary information to that provided by crystallography. Copyright © 2013 The Author. Published by Elsevier Inc. All rights reserved.

Regulation of plant plasma membrane H+- and Ca2+-ATPases by terminal domains

DEFF Research Database (Denmark)

Bækgaard, Lone; Fuglsang, Anja Thoe; Palmgren, Michael Gjedde

2005-01-01

In the last few years, major progress has been made to elucidate the structure, function, and regulation of P-type plasma membrane H(+)-and Ca(2+)-ATPases. Even though a number of regulatory proteins have been identified, many pieces are still lacking in order to understand the complete regulatory...... mechanisms of these pumps. In plant plasma membrane H(+)- and Ca(2+)-ATPases, autoinhibitory domains are situated in the C- and N-terminal domains, respectively. A model for a common mechanism of autoinhibition is discussed....

Role of post-translational modifications at the β-subunit ectodomain in complex association with a promiscuous plant P4-ATPase

DEFF Research Database (Denmark)

Costa, Sara; Marek, Magdalena; Axelsen, Kristian Buhl

2016-01-01

and can interact with several isoforms. In the present study, we used a site-directed mutagenesis approach to assess the role of post-translational modifications at the plant ALIS5 β-subunit ectodomain in the functionality of the promiscuous plant P4-ATPase ALA2. We identified two N-glycosylated residues......) compromises complex association, but the mutant β-subunits still promote complex trafficking and activity to some extent. In contrast, disruption of a conserved disulfide bond between Cys(158) and Cys(172) has no effect on the P4-ATPase complex. Our results demonstrate that post-translational modifications...

The Arabidopsis P4-ATPase ALA3 requires a ß-subunit to function in phospholipid translocation and secretory vesicle formation

DEFF Research Database (Denmark)

Lopez Marques, Rosa Laura

The Arabidopsis P4-ATPase ALA3 requires a ß-subunit to function in phospholipid translocation and secretory vesicle formation   Lisbeth R. Poulsen1, Rosa L. López-Marqués1, Stephen C. McDowell2, Juha Okkeri3, Dirk Licht3, Alexander Schulz1, Thomas Pomorski3,  Jeffrey F. Harper2, and Michael G....... Palmgren1 1Centre for Membrane Pumps in Cells and Disease - PUMPKIN, Danish National Research Foundation, Department of Plant Biology, University of Copenhagen, DK-1871 Frederiksberg C, Denmark 2Biochemistry Department MS200, University of Nevada Reno, NV 89557, USA 3Humboldt-University Berlin, Faculty...... and in inducing membrane curvature, which is a requirement for vesicle formation. We show that Aminophospholipid ATPase3 (ALA3), a member of the P4-ATPase subfamily in the plant Arabidopsis thaliana, localizes to the Golgi apparatus and that genetic lesions of ALA3 result in impaired growth of roots and shoots...

Cardioprotective effect of Sida rhomboidea. Roxb extract against isoproterenol induced myocardial necrosis in rats.

Science.gov (United States)

Thounaojam, Menaka C; Jadeja, Ravirajsinh N; Ansarullah; Karn, Sanjay S; Shah, Jigar D; Patel, Dipak K; Salunke, Sunita P; Padate, Geeta S; Devkar, Ranjitsinh V; Ramachandran, A V

2011-05-01

The present study investigates cardioprotective effect of Sida rhomboidea. Roxb (SR) extract on heart weight, plasma lipid profile, plasma marker enzymes, lipid peroxidation, endogenous enzymatic and non-enzymatic antioxidants and membrane bound ATPases against isoproterenol (IP) induced myocardial necrosis (MN) in rats. Rats treated with IP (85 mg/kg, s.c.) recorded significant (p<0.05) increment in heart weight, plasma lipid profile, plasma marker enzymes of cardiac damage, cardiac lipid peroxidation (LPO) and activity levels of Ca(+2) ATPase whereas there was significant (p<0.05) decrease in plasma HDL, cardiac endogenous enzymatic and non-enzymatic antioxidants, Na(+)-K(+) ATPase and Mg(+2) ATPase. Pre-treatment with SR extract (400 mg/kg per day, p.o.) for 30 consecutive days followed by IP injections on days 29th and 30th, showed significant (p<0.05) decrease in heart weight, plasma lipid profile, plasma marker enzymes of cardiac damage, cardiac lipid peroxidation, Ca(+2) ATPase and significant increase in plasma HDL, cardiac endogenous enzymatic and non-enzymatic antioxidants, Na(+)-K(+) ATPase and Mg(+2) ATPase compared to IP treated group. Hence, this study is the first scientific report on cardioprotective effect of SR against IP induced MN in rats. Copyright © 2010 Elsevier GmbH. All rights reserved.

Purification and functional motifs of the recombinant ATPase of orf virus.

Science.gov (United States)

Lin, Fong-Yuan; Chan, Kun-Wei; Wang, Chi-Young; Wong, Min-Liang; Hsu, Wei-Li

2011-10-01

Our previous study showed that the recombinant ATPase encoded by the A32L gene of orf virus displayed ATP hydrolysis activity as predicted from its amino acids sequence. This viral ATPase contains four known functional motifs (motifs I-IV) and a novel AYDG motif; they are essential for ATP hydrolysis reaction by binding ATP and magnesium ions. The motifs I and II correspond with the Walker A and B motifs of the typical ATPase, respectively. To examine the biochemical roles of these five conserved motifs, recombinant ATPases of five deletion mutants derived from the Taiping strain were expressed and purified. Their ATPase functions were assayed and compared with those of two wild type strains, Taiping and Nantou isolated in Taiwan. Our results showed that deletions at motifs I-III or IV exhibited lower activity than that of the wild type. Interestingly, deletion of AYDG motif decreased the ATPase activity more significantly than those of motifs I-IV deletions. Divalent ions such as magnesium and calcium were essential for ATPase activity. Moreover, our recombinant proteins of orf virus also demonstrated GTPase activity, though weaker than the original ATPase activity. Copyright © 2011 Elsevier Inc. All rights reserved.

Regulation of lipid droplet dynamics in Saccharomyces cerevisiae depends on the Rab7-like Ypt7p, HOPS complex and V1-ATPase

Directory of Open Access Journals (Sweden)

Isabelle Bouchez

2015-07-01

Full Text Available It has now been clearly shown that lipid droplets (LDs play a dynamic role in the cell. This was reinforced by LD proteomics which suggest that a significant number of trafficking proteins are associated with this organelle. Using microscopy, we showed that LDs partly co-localize with the vacuole in S. cerevisiae. Immunoblot experiments confirmed the association of the vacuolar Rab GTPase Rab7-like Ypt7p with LDs. We observed an increase in fatty acid content and LD number in ypt7Δ mutant and also changes in LD morphology and intra LD fusions, revealing a direct role for Ypt7p in LD dynamics. Using co-immunoprecipitation, we isolated potential Ypt7p partners including, Vma13p, the H subunit of the V1 part of the vacuolar (H+ ATPase (V-ATPase. Deletion of the VMA13 gene, as well as deletion of three other subunits of the V1 part of the V-ATPase, also increased the cell fatty acid content and LD number. Mutants of the Homotypic fusion and vacuole protein sorting (HOPS complex showed similar phenotypes. Here, we demonstrated that LD dynamics and membrane trafficking between the vacuole and LDs are regulated by the Rab7-like Ypt7p and are impaired when the HOPS complex and the V1 domain of the V-ATPase are defective.

Cardiac effects of 3 months treatment of acromegaly evaluated by magnetic resonance imaging and B-type natriuretic peptides

DEFF Research Database (Denmark)

Andreassen, Mikkel; Faber, Jens; Kjær, Andreas

2010-01-01

Long-term treatment of acromegaly prevents aggravation and reverses associated heart disease. A previous study has shown a temporary increase in serum levels of the N-terminal fraction of pro B-type natriuretic peptide (NT-proBNP) suggesting an initial decline in cardiac function when treatment...... of acromegaly is initiated. This was a three months prospective study investigating short-term cardiac effects of treatment in acromegalic patients. Cardiac function was evaluated by the gold standard method cardiac magnetic resonance imaging (CMRI) and circulating levels of B-type natriuretic peptides (BNP......) (95% CI 3-14), P = 0.007) and an increase in levels of BNP (median (ranges) 7 (0.58-286) vs. 20 (1-489) pg/mL, P = 0.033) and of NT-proBNP (63 (20-1004) vs. 80 (20-3391) pg/mL, P = 0.027). Assessed by the highly sensitive and precise CMRI method, 3 months treatment of acromegaly resulted...

Selective induction of high-ouabain-affinity isoform of Na+-K+-ATPase by thyroid hormone

International Nuclear Information System (INIS)

Haber, R.S.; Loeb, J.N.

1988-01-01

The administration of thyroid hormone is known to result in an induction of the Na + -K + -adenosinetriphosphatase (Na + -K + -ATPase) in rat skeletal muscle and other thyroid hormone-responsive tissues. Since the Na + -K + -ATPase in a variety of mammalian tissues has recently been reported to exist in at least two forms distinguishable by differing affinities for the inhibitory cardiac glycoside ouabain. The authors have studied the effects of 3,3',5-triiodo-L-thyronine (T 3 ) treatment on these two forms of the enzyme in rat diaphragm. The inhibition of Na + -K + -ATPase activity in a crude membrane fraction by varying concentrations of ouabain conformed to a biphasic pattern consistent with the presence of two distinct isoforms with inhibition constants (K I s) for ouabain of ∼10 -7 and 10 -4 M, respectively. Measurement of the specific binding of [ 3 H]ouabain to these membranes confirmed the presence of a class of high-affinity ouabain binding sites with a dissociation constant (K d ) of slightly less than 10 -7 M, whose maximal binding capacity was increased by T 3 treatment by 185%. Binding studies in unfractionated homogenates of diaphragm similarly demonstrated the presence of high-affinity sites whose maximal binding capacity was increased by T 3 treatment. Quantitation of both the high- and low-ouabain-affinity forms of the Na + -K + -ATPase by ouabain-dependent phosphorylation from [ 32 P]orthophosphate confirmed that T 3 treatment markedly increased the number of high-affinity sites while having little effect on the number of low-affinity sites. These observations provide, to our knowledge, the first demonstration that these two forms of the Na + -K + -ATPase are subject to selective hormonal induction

Kinetics of the flash-induced P515 response in relation to the H+-permeability of the membrane bound ATPase in spinach chloroplasts

Energy Technology Data Exchange (ETDEWEB)

Peters, R.L.; van Kooten, O.; Vredenberg, W.J.

1985-08-01

The effect of dicyclohexylcarbodiimide (DCCD) on the kinetics of the flash-induced P515 response and on the activity of the ATPase was investigated in isolated spinach chloroplasts. It was found that after the addition of 5 X 10(-8)mol DCCD the rate of ATP hydrolysis induced by a period of 60 sec illumination was decreased to less than 5% of its original value. At this concentration, hardly any effect, if at all, could be detected on the kinetics of the flash-induced P515 response, neither in dark-adapted nor in light-activated chloroplasts. It was concluded that the presence of concentrations of DCCD, sufficiently high to affect the ATPase activity, does not affect the kinetics of the flash-induced P515 response. Since DCCD decreases the H+ permeability of the membrane-bound ATPase, it was concluded that this permeability coefficient for protons is not an important factor in the regulation of the flash-induced membrane potential and, therefore, does not affect the kinetics of the flash-induced P515 response.

Regulation of branchial V-H(+)-ATPase, Na(+)/K(+)-ATPase and NHE2 in response to acid and base infusions in the Pacific spiny dogfish (Squalus acanthias).

Science.gov (United States)

Tresguerres, Martin; Katoh, Fumi; Fenton, Heather; Jasinska, Edyta; Goss, Greg G

2005-01-01

To study the mechanisms of branchial acid-base regulation, Pacific spiny dogfish were infused intravenously for 24 h with either HCl (495+/- 79 micromol kg(-1) h(-1)) or NaHCO(3) (981+/-235 micromol kg(-1) h(-1)). Infusion of HCl produced a transient reduction in blood pH. Despite continued infusion of acid, pH returned to normal by 12 h. Infusion of NaHCO(3) resulted in a new steady-state acid-base status at approximately 0.3 pH units higher than the controls. Immunostained serial sections of gill revealed the presence of separate vacuolar proton ATPase (V-H(+)-ATPase)-rich or sodium-potassium ATPase (Na(+)/K(+)-ATPase)-rich cells in all fish examined. A minority of the cells also labeled positive for both transporters. Gill cell membranes prepared from NaHCO(3)-infused fish showed significant increases in both V-H(+)-ATPase abundance (300+/-81%) and activity. In addition, we found that V-H(+)-ATPase subcellular localization was mainly cytoplasmic in control and HCl-infused fish, while NaHCO(3)-infused fish demonstrated a distinctly basolateral staining pattern. Western analysis in gill membranes from HCl-infused fish also revealed increased abundance of Na(+)/H(+) exchanger 2 (213+/-5%) and Na(+)/K(+)-ATPase (315+/-88%) compared to the control.

Cardiac Iodine-123 metaiodobenzylguanidine (123I-MIBG) scintigraphy parameter predicts cardiac and cerebrovascular events in type 2 diabetic patients without structural heart disease

International Nuclear Information System (INIS)

Yufu, Kunio; Takahashi, Naohiko; Okada, Norihiro; Shinohara, Tetsuji; Nakagawa, Mikiko; Hara, Masahide; Yoshimatsu, Hironobu; Saikawa, Tetsunori

2012-01-01

Cardiac iodine-123 metaiodobenzylguanidine ( 123 I-MIBG) scintigraphy is an established method of assessment of cardiovascular sympathetic function. The aim of the present study was to investigate the long-term cardiovascular predictive value of cardiac 123 I-MIBG scintigraphy parameters in Japanese type 2 diabetic patients without structural heart disease. Cardiac 123 I-MIBG scintigraphy in 108 patients with type 2 diabetes who did not have structural heart disease, was evaluated. The washout rate (WR) was considered enhanced if it was ≥40%. Accurate follow-up information for 4.6 years was obtained in 54 enhanced WR patients (27 male; mean age, 61±11 years) and in 54 sex- and age-matched preserved WR patients (27 male; mean age, 61±10 years). Major adverse cardiac and cerebrovascular events (MACCE) were investigated. During follow-up, 10 enhanced WR patients developed MACCE including cardiac death, coronary revascularization, stroke, and congestive heart failure, while MACCE occurred in only 3 male patients. The Kaplan-Meier curves indicated that enhanced WR patients had higher incidence of MACCE than those with preserved WR (P 123 I-MIBG scintigraphy at baseline has long-term cardiovascular predictive value in Japanese patients with type 2 diabetes without structural heart disease. (author)

Quantitative measurement of membrane Na+-K+ ATPase activity using thallium-201: comparison with rubidium-86

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Lee, Jae Tae; Shon, Sang Kyun; Lee, Kyu Bo; Lee, In Kyu

1998-01-01

Na + -K + ATPase activity has been estimated by the degree of inhibition of cation transport by cardiac glycosides (ouabain) using Rb-86 as a substrate. The biological characteristics of Tl-201 is known to be similar to those of potassium as a transport substrate in the presence of glucose, insulin or phobol myristate acetate (PMA). The purpose of this study was to measure ouabain sensitive Na + -K + ATPase activity using Tl-201 and compare with that using Rb-86. Smooth muscle cells isolated from rat aorta or human placental umbilical artery were cultured, and used to measure cellular Na + -K + ATPase activity. Na + -K + ATPase activity was measured as a percentage decrease in cellular uptake of Tl-201 or Rb-86 by ouabain under the presence of glucose, insulin or PMA in media. Na + -K + ATPase activity measured with Tl-201, as a transport substrate, was not different from those measured with Rb-86 in rat or human smooth muscle cell preparation. Incubation with high concentration glucose resulted in about 30% decrease in enzyme activity. In contrast, insulin or PMA resulted in 50-70% or 28% increase from baseline activity, respectively. These results suggests that Tl-201 could replace Rb-86 in measurement of ouabain sensitive Na + -K + ATPase activity in vitro. High level of glucose concentration decreased cellular Na + -K + ATPase activity, but insulin or PMA increased it

A novel calmodulin-regulated Ca2+-ATPase (ACA2) from Arabidopsis with an N-terminal autoinhibitory domain

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Harper, J. F.; Hong, B.; Hwang, I.; Guo, H. Q.; Stoddard, R.; Huang, J. F.; Palmgren, M. G.; Sze, H.; Evans, M. L. (Principal Investigator)

1998-01-01

To study transporters involved in regulating intracellular Ca2+, we isolated a full-length cDNA encoding a Ca2+-ATPase from a model plant, Arabidopsis, and named it ACA2 (Arabidopsis Ca2+-ATPase, isoform 2). ACA2p is most similar to a "plasma membrane-type" Ca2+-ATPase, but is smaller (110 kDa), contains a unique N-terminal domain, and is missing a long C-terminal calmodulin-binding regulatory domain. In addition, ACA2p is localized to an endomembrane system and not the plasma membrane, as shown by aqueous-two phase fractionation of microsomal membranes. ACA2p was expressed in yeast as both a full-length protein (ACA2-1p) and an N-terminal truncation mutant (ACA2-2p; Delta residues 2-80). Only the truncation mutant restored the growth on Ca2+-depleted medium of a yeast mutant defective in both endogenous Ca2+ pumps, PMR1 and PMC1. Although basal Ca2+-ATPase activity of the full-length protein was low, it was stimulated 5-fold by calmodulin (50% activation around 30 nM). In contrast, the truncated pump was fully active and insensitive to calmodulin. A calmodulin-binding sequence was identified within the first 36 residues of the N-terminal domain, as shown by calmodulin gel overlays on fusion proteins. Thus, ACA2 encodes a novel calmodulin-regulated Ca2+-ATPase distinguished by a unique N-terminal regulatory domain and a non-plasma membrane localization.

Cardiac lipid content is unresponsive to a physical activity training intervention in type 2 diabetic patients, despite improved ejection fraction

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Leiner Tim

2011-05-01

Full Text Available Abstract Background Increased cardiac lipid content has been associated with diabetic cardiomyopathy. We recently showed that cardiac lipid content is reduced after 12 weeks of physical activity training in healthy overweight subjects. The beneficial effect of exercise training on cardiovascular risk is well established and the decrease in cardiac lipid content with exercise training in healthy overweight subjects was accompanied by improved ejection fraction. It is yet unclear whether diabetic patients respond similarly to physical activity training and whether a lowered lipid content in the heart is necessary for improvements in cardiac function. Here, we investigated whether exercise training is able to lower cardiac lipid content and improve cardiac function in type 2 diabetic patients. Methods Eleven overweight-to-obese male patients with type 2 diabetes mellitus (age: 58.4 ± 0.9 years, BMI: 29.9 ± 0.01 kg/m2 followed a 12-week training program (combination endurance/strength training, three sessions/week. Before and after training, maximal whole body oxygen uptake (VO2max and insulin sensitivity (by hyperinsulinemic, euglycemic clamp was determined. Systolic function was determined under resting conditions by CINE-MRI and cardiac lipid content in the septum of the heart by Proton Magnetic Resonance Spectroscopy. Results VO2max increased (from 27.1 ± 1.5 to 30.1 ± 1.6 ml/min/kg, p = 0.001 and insulin sensitivity improved upon training (insulin stimulated glucose disposal (delta Rd of glucose improved from 5.8 ± 1.9 to 10.3 ± 2.0 μmol/kg/min, p = 0.02. Left-ventricular ejection fraction improved after training (from 50.5 ± 2.0 to 55.6 ± 1.5%, p = 0.01 as well as cardiac index and cardiac output. Unexpectedly, cardiac lipid content in the septum remained unchanged (from 0.80 ± 0.22% to 0.95 ± 0.21%, p = 0.15. Conclusions Twelve weeks of progressive endurance/strength training was effective in improving VO2max, insulin sensitivity

Proteasome Inhibition Contributed to the Cytotoxicity of Arenobufagin after Its Binding with Na, K-ATPase in Human Cervical Carcinoma HeLa Cells.

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Yue, Qingxi; Zhen, Hong; Huang, Ming; Zheng, Xi; Feng, Lixing; Jiang, Baohong; Yang, Min; Wu, Wanying; Liu, Xuan; Guo, Dean

2016-01-01

Although the possibility of developing cardiac steroids/cardiac glycosides as novel cancer therapeutic agents has been recognized, the mechanism of their anticancer activity is still not clear enough. Toad venom extract containing bufadienolides, which belong to cardiac steroids, has actually long been used as traditional Chinese medicine in clinic for cancer therapy in China. The cytotoxicity of arenobufagin, a bufadienolide isolated from toad venom, on human cervical carcinoma HeLa cells was checked. And, the protein expression profile of control HeLa cells and HeLa cells treated with arenobufagin for 48 h was analyzed using two-dimensional electrophoresis, respectively. Differently expressed proteins in HeLa cells treated with arenobufagin were identified and the pathways related to these proteins were mapped from KEGG database. Computational molecular docking was performed to verify the binding of arenobufagin and Na, K-ATPase. The effects of arenobufagin on Na, K-ATPase activity and proteasome activity of HeLa cells were checked. The protein-protein interaction network between Na, K-ATPase and proteasome was constructed and the expression of possible intermediate proteins ataxin-1 and translationally-controlled tumor protein in HeLa cells treated with arenobufagin was then checked. Arenobufagin induced apoptosis and G2/M cell cycle arrest in HeLa cells. The cytotoxic effect of arenobufagin was associated with 25 differently expressed proteins including proteasome-related proteins, calcium ion binding-related proteins, oxidative stress-related proteins, metabolism-related enzymes and others. The results of computational molecular docking revealed that arenobufagin was bound in the cavity formed by the transmembrane alpha subunits of Na, K-ATPase, which blocked the pathway of extracellular Na+/K+ cation exchange and inhibited the function of ion exchange. Arenobufagin inhibited the activity of Na, K-ATPase and proteasome, decreased the expression of Na, K-ATPase

HDAC Inhibition Improves the Sarcoendoplasmic Reticulum Ca2+-ATPase Activity in Cardiac Myocytes.

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Meraviglia, Viviana; Bocchi, Leonardo; Sacchetto, Roberta; Florio, Maria Cristina; Motta, Benedetta M; Corti, Corrado; Weichenberger, Christian X; Savi, Monia; D'Elia, Yuri; Rosato-Siri, Marcelo D; Suffredini, Silvia; Piubelli, Chiara; Pompilio, Giulio; Pramstaller, Peter P; Domingues, Francisco S; Stilli, Donatella; Rossini, Alessandra

2018-01-31

SERCA2a is the Ca 2+ ATPase playing the major contribution in cardiomyocyte (CM) calcium removal. Its activity can be regulated by both modulatory proteins and several post-translational modifications. The aim of the present work was to investigate whether the function of SERCA2 can be modulated by treating CMs with the histone deacetylase (HDAC) inhibitor suberanilohydroxamic acid (SAHA). The incubation with SAHA (2.5 µM, 90 min) of CMs isolated from rat adult hearts resulted in an increase of SERCA2 acetylation level and improved ATPase activity. This was associated with a significant improvement of calcium transient recovery time and cell contractility. Previous reports have identified K464 as an acetylation site in human SERCA2. Mutants were generated where K464 was substituted with glutamine (Q) or arginine (R), mimicking constitutive acetylation or deacetylation, respectively. The K464Q mutation ameliorated ATPase activity and calcium transient recovery time, thus indicating that constitutive K464 acetylation has a positive impact on human SERCA2a (hSERCA2a) function. In conclusion, SAHA induced deacetylation inhibition had a positive impact on CM calcium handling, that, at least in part, was due to improved SERCA2 activity. This observation can provide the basis for the development of novel pharmacological approaches to ameliorate SERCA2 efficiency.

MxiN Differentially Regulates Monomeric and Oligomeric Species of the Shigella Type Three Secretion System ATPase Spa47.

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Case, Heather B; Dickenson, Nicholas E

2018-04-17

Shigella rely entirely on the action of a single type three secretion system (T3SS) to support cellular invasion of colonic epithelial cells and to circumvent host immune responses. The ATPase Spa47 resides at the base of the Shigella needle-like type three secretion apparatus (T3SA), supporting protein secretion through the apparatus and providing a likely means for native virulence regulation by Shigella and a much needed target for non-antibiotic therapeutics to treat Shigella infections. Here, we show that MxiN is a differential regulator of Spa47 and that its regulatory impact is determined by the oligomeric state of the Spa47 ATPase, with which it interacts. In vitro and in vivo characterization shows that interaction of MxiN with Spa47 requires the six N-terminal residues of Spa47 that are also necessary for stable Spa47 oligomer formation and activation. This interaction with MxiN negatively influences the activity of Spa47 oligomers while upregulating the ATPase activity of monomeric Spa47. Detailed kinetic analyses of monomeric and oligomeric Spa47 in the presence and absence of MxiN uncover additional mechanistic insights into the regulation of Spa47 by MxiN, suggesting that the MxiN/Spa47 species resulting from interaction with monomeric and oligomeric Spa47 are functionally distinct and that both could be involved in Shigella T3SS regulation. Uncovering regulation of Spa47 by MxiN addresses an important gap in the current understanding of how Shigella controls T3SA activity and provides the first description of differential T3SS ATPase regulation by a native T3SS protein.

Epigallocatechin-3-Gallate Protects Erythrocyte Ca2+-ATPase and Na+/K+-ATPase Against Oxidative Induced Damage During Aging in Humans

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Prabhanshu Kumar

2014-10-01

Full Text Available Purpose: The main purpose of this study was to investigate the protective role of epigallocatechin-3-gallate on tertiary butyl hydroperoxide induced oxidative damage in erythrocyte during aging in humans. Methods: Human erythrocyte membrane bound Ca2+-ATPase and Na+/K+-ATPase activities were determined as a function of human age. Protective role of epigallocatechin-3-gallate was evaluated by in vitro experiments by adding epigallocatechin-3-gallate in concentration dependent manner (final concentration range 10-7M to 10-4M to the enzyme assay medium. Oxidative stress was induced in vitro by incubating washed erythrocyte ghosts with tertiary butyl hydroperoxide (10-5 M final concentration. Results: We have reported concentration dependent effect of epigallocatechin-3-gallate on tertiary butyl hydroperoxide induced damage on activities of Ca2+-ATPase and Na+/K+-ATPase during aging in humans. We have detected a significant (p < 0.001 decreased activity of Ca2+-ATPase and Na+/K+ -ATPase as a function of human age. Epigallocatechin-3-gallate protected ATPases against tertiary butyl hydroperoxide induced damage in concentration dependent manner during aging in humans. Conclusion: Epigallocatechin-3-gallate is a powerful antioxidant that is capable of protecting erythrocyte Ca2+-ATPase and Na+/K+ -ATPase against oxidative stress during aging in humans. We may propose hypothesis that a high intake of catechin rich diet may provide some protection against development of aging and age related diseases.

Type D personality is associated with impaired psychological status and unhealthy lifestyle in Icelandic cardiac patients: A cross-sectional study

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Svansdottir Erla

2012-01-01

Full Text Available Abstract Background Type D (distressed personality has been associated with adverse cardiac prognosis and poor emotional well-being in cardiac patients, but it is still unclear what mechanisms link Type D personality with poor clinical outcomes in cardiac patients. In the present cohort of Icelandic cardiac patients, we examined potential pathways that may explain this relationship. The objectives were to examine 1 the association between Type D personality and impaired psychological status, and to explore whether this association is independent of disease severity; and 2 the association between Type D personality and an unhealthy lifestyle. Methods A sample of 268 Icelandic coronary angiography patients (74% males (N = 199; mean age 62.9 years (SD 10.5, range 28-85 years completed the Type D Scale (DS14, Hospital Anxiety and Depression Scale (HADS, and Perceived Stress Scale (PSS at hospitalization. Health-related behaviors were assessed 4 months following angiography. Clinical data were collected from medical files. Results Type D personality was associated with an increased risk of anxiety (OR 2.97, 95% CI:1.55-5.69, depression (OR 4.01, 95% CI:1.42-11.29, and stress (OR 5.99, 95% CI:3.08-11.63, independent of demographic variables and disease severity. Furthermore, fish consumption was lower among Type Ds, as 21% of Type Ds versus 5% of non-Type Ds consumed fish p p = 0.024 and to use antidepressants (17% versus 9%, p = 0.049 and sleeping pills (49% versus 33%, p = 0.019 compared to non-Type Ds. Type D personality was not associated with other health-related behaviors, aside from trends towards less fruit and vegetable consumption, and more weight gain. Conclusion Type D personality was associated with psychological distress and an unhealthy lifestyle in Icelandic cardiac patients. Future studies should further investigate the association between Type D personality and health-related behaviors.

V-ATPase is a candidate therapeutic target for Ewing sarcoma.

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Avnet, Sofia; Di Pompo, Gemma; Lemma, Silvia; Salerno, Manuela; Perut, Francesca; Bonuccelli, Gloria; Granchi, Donatella; Zini, Nicoletta; Baldini, Nicola

2013-08-01

Suppression of oxidative phosphorylation combined with enhanced aerobic glycolysis and the resulting increased generation of protons are common features of several types of cancer. An efficient mechanism to escape cell death resulting from intracellular acidification is proton pump activation. In Ewing sarcoma (ES), although the tumor-associated chimeric gene EWS-FLI1 is known to induce the accumulation of hypoxia-induced transcription factor HIF-1α, derangements in metabolic pathways have been neglected so far as candidate pathogenetic mechanisms. In this paper, we observed that ES cells simultaneously activate mitochondrial respiration and high levels of glycolysis. Moreover, although the most effective detoxification mechanism of proton intracellular storage is lysosomal compartmentalization, ES cells show a poorly represented lysosomal compartment, but a high sensitivity to the anti-lysosomal agent bafilomycin A1, targeting the V-ATPase proton pump. We therefore investigated the role of V-ATPase in the acidification activity of ES cells. ES cells with the highest GAPDH and V-ATPase expression also showed the highest acidification rate. Moreover, the localization of V-ATPase was both on the vacuolar and the plasma membrane of all ES cell lines. The acidic extracellular pH that we reproduced in vitro promoted high invasion ability and clonogenic efficiency. Finally, targeting V-ATPase with siRNA and omeprazole treatments, we obtained a significant selective reduction of tumor cell number. In summary, glycolytic activity and activation of V-ATPase are crucial mechanisms of survival of ES cells and can be considered as promising selective targets for the treatment of this tumor. Copyright © 2013 Elsevier B.V. All rights reserved.

Unique type of isolated cardiac valvular amyloidosis

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Reehana Salma

2006-10-01

Full Text Available Abstract Background Amyloid deposition in heart is a common occurrence in systemic amyloidosis. But localised valvular amyloid deposits are very uncommon. It was only in 1922 that the cases of valvular amyloidosis were reported. Then in 1980, Goffin et al reported another type of valvular amyloidosis, which he called the dystrophic valvular amyloidosis. We report a case of aortic valve amyloidosis which is different from the yet described valvular amyloidosis. Case presentation A 72 years old gentleman underwent urgent aortic valve replacement. Intraoperatively, a lesion was found attached to the inferior surface of his bicuspid aortic valve. Histopathology examination of the valve revealed that the lesion contained amyloid deposits, identified as AL amyloidosis. The serum amyloid A protein (SAP scan was normal and showed no evidence of systemic amyloidosis. The ECG and echocardiogram were not consistent with cardiac amyloidosis. Conclusion Two major types of cardiac amyloidosis have been described in literature: primary-myelomatous type (occurs with systemic amyolidosis, and senile type(s. Recently, a localised cardiac dystrophic valvular amyloidosis has been described. In all previously reported cases, there was a strong association of localised valvular amyloidosis with calcific deposits. Ours is a unique case which differs from the previously reported cases of localised valvular amyloidosis. In this case, the lesion was not associated with any scar tissue. Also there was no calcific deposit found. This may well be a yet unknown type of isolated valvular amyloidosis.

Induction of Heavy-Metal-Transporting CPX-Type ATPases during Acid Adaptation in Lactobacillus bulgaricus▿

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Penaud, S.; Fernandez, A.; Boudebbouze, S.; Ehrlich, S. D.; Maguin, E.; van de Guchte, M.

2006-01-01

Lactobacillus bulgaricus is a lactic acid bacteria (LAB) that, through the production of lactic acid, gradually acidifies its environment during growth. In the course of this process, L. bulgaricus acquires an improved tolerance to acidity. A survey of the recently established genome sequence shows that this bacterium possesses few of the pH control functions that have been described in other LAB and raises the question of what other mechanisms could be involved in its adaptation to the decreasing environmental pH. In some bacteria other than LAB, ion transport systems have been implicated in acid adaptation. We therefore studied the expression of this type of transport system during acid adaptation in L. bulgaricus by reverse transcription and real-time quantitative PCR and mapped transcription start sites. Intriguingly, the most significantly induced were three ATPases carrying the CPX signature of heavy-metal transporters. Protein homology and the presence of a conserved sequence motif in the promoter regions of the genes encoding these proteins strongly suggest that they are involved in copper homeostasis. Induction of this system is thought to assist in avoiding indirect damage that could result from medium acidification. PMID:16997986

Estrogen deprivation aggravates cardiac hypertrophy in nonobese Type 2 diabetic Goto-Kakizaki (GK) rats.

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Apaijai, Nattayaporn; Charoenphandhu, Narattaphol; Ittichaichareon, Jitjiroj; Suntornsaratoon, Panan; Krishnamra, Nateetip; Aeimlapa, Ratchaneevan; Chattipakorn, Siriporn C; Chattipakorn, Nipon

2017-10-31

Both Type 2 diabetes mellitus (T2DM) and estrogen deprivation have been shown to be associated with the development of cardiovascular disease and adverse cardiac remodeling. However, the role of estrogen deprivation on adverse cardiac remodeling in nonobese T2DM rats has not been clearly elucidated. We hypothesized that estrogen-deprivation aggravates adverse cardiac remodeling in Goto-Kakizaki (GK) rats. Wild-type (WT) and GK rats at the age of 9 months old were divided into two subgroups to have either a sham operation (WTS, GKS) or a bilateral ovariectomy (WTO, GKO) ( n = 6/subgroup). Four months after the operation, the rats were killed, and the heart was excised rapidly. Metabolic parameters, cardiomyocytes hypertrophy, cardiac fibrosis, and biochemical parameters were determined. GK rats had hyperglycemia with hypoinsulinemia, and estrogen deprivation did not increase the severity of T2DM. Cardiac hypertrophy, cardiac oxidative stress, and phosphor-antinuclear factor κB were higher in WTO and GKS rats than WTS rats, and they markedly increased in GKO rats compared with GKS rats. Furthermore, cardiac fibrosis, transforming growth factor-β, Bax, phosphor-p38, and peroxisome proliferator- activated receptor γ coactivator-1α expression were increased in GKS and GKO rats compared with the lean rats. However, mitochondrial dynamics proteins including dynamin-related protein 1 and mitofusin-2 were not altered by T2DM and estrogen deprivation. Although estrogen deprivation did not aggravate T2DM in GK rats, it increased the severity of cardiac hypertrophy by provoking cardiac inflammation and oxidative stress in nonobese GK rats. © 2017 The Author(s).

Adaptation of H+-pumping and plasma membrane H+ ATPase activity in proteoid roots of white lupin under phosphate deficiency.

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Yan, Feng; Zhu, Yiyong; Müller, Caroline; Zörb, Christian; Schubert, Sven

2002-05-01

White lupin (Lupinus albus) is able to adapt to phosphorus deficiency by producing proteoid roots that release a huge amount of organic acids, resulting in mobilization of sparingly soluble soil phosphate in rhizosphere. The mechanisms responsible for the release of organic acids by proteoid root cells, especially the trans-membrane transport processes, have not been elucidated. Because of high cytosolic pH, the release of undissociated organic acids is not probable. In the present study, we focused on H+ export by plasma membrane H+ ATPase in active proteoid roots. In vivo, rhizosphere acidification of active proteoid roots was vanadate sensitive. Plasma membranes were isolated from proteoid roots and lateral roots from P-deficient and -sufficient plants. In vitro, in comparison with two types of lateral roots and proteoid roots of P-sufficient plants, the following increase of the various parameters was induced in active proteoid roots of P-deficient plants: (a) hydrolytic ATPase activity, (b) Vmax and Km, (c) H+ ATPase enzyme concentration of plasma membrane, (d) H+-pumping activity, (e) pH gradient across the membrane of plasmalemma vesicles, and (f) passive H+ permeability of plasma membrane. In addition, lower vanadate sensitivity and more acidic pH optimum were determined for plasma membrane ATPase of active proteoid roots. Our data support the hypothesis that in active proteoid root cells, H+ and organic anions are exported separately, and that modification of plasma membrane H+ ATPase is essential for enhanced rhizosphere acidification by active proteoid roots.

Identification of a mutant α1 Na/K-ATPase that pumps but is defective in signal transduction.

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Lai, Fangfang; Madan, Namrata; Ye, Qiqi; Duan, Qiming; Li, Zhichuan; Wang, Shaomeng; Si, Shuyi; Xie, Zijian

2013-05-10

It has not been possible to study the pumping and signaling functions of Na/K-ATPase independently in live cells. Both cell-free and cell-based assays indicate that the A420P mutation abolishes the Src regulatory function of Na/K-ATPase. A420P mutant has normal pumping but not signaling function. Identification of Src regulation-null mutants is crucial for addressing physiological role of Na/K-ATPase. The α1 Na/K-ATPase possesses both pumping and signaling functions. However, it has not been possible to study these functions independently in live cells. We have identified a 20-amino acid peptide (Ser-415 to Gln-434) (NaKtide) from the nucleotide binding domain of α1 Na/K-ATPase that binds and inhibits Src in vitro. The N terminus of NaKtide adapts a helical structure. In vitro kinase assays showed that replacement of residues that contain a bulky side chain in the helical structure of NaKtide by alanine abolished the inhibitory effect of the peptide on Src. Similarly, disruption of helical structure by proline replacement, either single or in combination, reduced the inhibitory potency of NaKtide on Src. To identify mutant α1 that retains normal pumping function but is defective in Src regulation, we transfected Na/K-ATPase α1 knockdown PY-17 cells with expression vectors of wild type or mutant α1 carrying Ala to Pro mutations in the region of NaKtide helical structure and generated several stable cell lines. We found that expression of either A416P or A420P or A425P mutant fully restored the α1 content and consequently the pumping capacity of cells. However, in contrast to A416P, either A420P or A425P mutant was incapable of interacting and regulating cellular Src. Consequently, expression of these two mutants caused significant inhibition of ouabain-activated signal transduction and cell growth. Thus we have identified α1 mutant that has normal pumping function but is defective in signal transduction.

Selective induction of high-ouabain-affinity isoform of Na sup + -K sup + -ATPase by thyroid hormone

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Haber, R.S.; Loeb, J.N. (Columbia Univ., New York, NY (USA))

1988-12-01

The administration of thyroid hormone is known to result in an induction of the Na{sup +}-K{sup +}-adenosinetriphosphatase (Na{sup +}-K{sup +}-ATPase) in rat skeletal muscle and other thyroid hormone-responsive tissues. Since the Na{sup +}-K{sup +}-ATPase in a variety of mammalian tissues has recently been reported to exist in at least two forms distinguishable by differing affinities for the inhibitory cardiac glycoside ouabain. The authors have studied the effects of 3,3{prime},5-triiodo-L-thyronine (T{sub 3}) treatment on these two forms of the enzyme in rat diaphragm. The inhibition of Na{sup +}-K{sup +}-ATPase activity in a crude membrane fraction by varying concentrations of ouabain conformed to a biphasic pattern consistent with the presence of two distinct isoforms with inhibition constants (K{sub I}s) for ouabain of {approximately}10{sup {minus}7} and 10{sup {minus}4} M, respectively. Measurement of the specific binding of ({sup 3}H)ouabain to these membranes confirmed the presence of a class of high-affinity ouabain binding sites with a dissociation constant (K{sub d}) of slightly less than 10{sup {minus}7}M, whose maximal binding capacity was increased by T{sub 3} treatment by 185%. Binding studies in unfractionated homogenates of diaphragm similarly demonstrated the presence of high-affinity sites whose maximal binding capacity was increased by T{sub 3} treatment. Quantitation of both the high- and low-ouabain-affinity forms of the Na{sup +}-K{sup +}-ATPase by ouabain-dependent phosphorylation from ({sup 32}P)orthophosphate confirmed that T{sub 3} treatment markedly increased the number of high-affinity sites while having little effect on the number of low-affinity sites. These observations provide, to our knowledge, the first demonstration that these two forms of the Na{sup +}-K{sup +}-ATPase are subject to selective hormonal induction.

Milrinone and thyroid hormone stimulate myocardial membrane Ca2+-ATPase activity and share structural homologies.

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Mylotte, K M; Cody, V; Davis, P J; Davis, F B; Blas, S D; Schoenl, M

1985-01-01

We have recently shown that thyroid hormone in physiological concentrations stimulates sarcolemma-enriched rabbit-myocardial-membrane Ca2+-ATPase in vitro. In this study, milrinone [2-methyl-5-cyano-(3,4'-bipyridin)-6(1H)-one], a cardiac inotropic agent, was thyromimetic in the same system. At clinically achievable concentrations (50-500 nM), milrinone significantly stimulated membrane Ca2+-ATPase in vitro. This action was antagonized by W-7 [N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide], an agent that also blocks thyroid hormone action on the Ca2+-ATPase, at concentrations as low as 5 microM. Progressive additions of milrinone to membranes incubated with a fixed concentration of thyroxine (0.10 nM) or triiodothyronine resulted in a progressive obliteration of the thyroid hormone effect on Ca2+-ATPase. Amrinone [5-amino-(3,4'-bipyridin)-6(1H)-one], the parent bipyridine of milrinone, had no effect on myocardial Ca2+-ATPase activity. X-ray crystallographic analysis of milrinone and amrinone revealed structural homologies between the phenolic ring of thyroxine and the substituted ring of milrinone, whereas amrinone did not share these homologies. The mechanism(s) of the inotropic actions of thyroxine and of milrinone is not clearly understood, but these observations implicate Ca2+-ATPase, a calcium pump-associated enzyme, as one mediator of the effects on the heart of these two compounds. PMID:2933747

Effect of TGFβ on Na+/K+ ATPase activity in megakaryocytes

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Hosseinzadeh, Zohreh; Schmid, Evi; Shumilina, Ekaterina; Laufer, Stefan; Borst, Oliver; Gawaz, Meinrad; Lang, Florian

2014-01-01

Highlights: • TGFß1 markedly up-regulates Na + /K + ATPase in megakaryocytes. • The effect is abrogated by p38-MAP kinase inhibitor skepinone. • The effect is abrogated by SGK inhibitor EMD638683. • The effect is abrogated by NF-κB inhibitor wogonin. - Abstract: The Na + /K + ATPase generates the Na + and K + concentration gradients across the plasma membrane and is thus essential for cellular electrolyte homeostasis, cell membrane potential and cell volume maintenance. A powerful regulator of Na + /K + ATPase is the serum- and glucocorticoid-inducible kinase 1 (SGK1). The most powerful known regulator of SGK1 expression is TGFß1, which is pivotal in the regulation of megakaryocyte maturation and platelet formation. Signaling involved in the upregulation of SGK1 by TGFß1 includes p38 mitogen activated protein (MAP) kinase. SGK1 in turn phosphorylates the IκB kinase (IKKα/β), which phosphorylates the inhibitor protein IκBα thus triggering nuclear translocation of nuclear factor kappa B (NF-κB). The present study explored whether TGFβ influences Na + /K + ATPase activity in megakaryocytes, and if so, whether the effect of TGß1 requires p38 MAP kinase, SGK1 and/or NF-κB. To this end, murine megakaryocytes were treated with TGFß1 and Na + /K + ATPase activity determined from K + induced current utilizing whole cell patch clamp. The pump current (I pump ) was determined in the absence and presence of Na + /K + ATPase inhibitor ouabain (100 μM). TGFß1 (60 ng/ml) was added in the absence or presence of p38 MAP kinase inhibitor skepinone-L (1 μM), SGK1 inhibitor EMD638683 (50 μM) or NF-κB inhibitor wogonin (50 nM). As a result, the I pump was significantly increased by pretreatment of the megakaryocytes with TGFß1, an effect reaching statistical significance within 16 and 24 h and virtually abrogated in the presence of skepinone-L, EMD638683 or wogonin. In conclusion, TGFß1 is a powerful regulator of megakaryocytic Na + /K + ATPase activity

Biochemical characterization of the plasma membrane H+ - ATPase from red beet (Beta vulgaris) hypocotyl tissue

International Nuclear Information System (INIS)

Oleski, N.A.

1986-01-01

Several biochemical techniques including selective solubilization followed by gel filtration or various types of affinity chromatography, and antibody production were employed in an attempt to purify the plasma membrane H + - ATPase from red beet hypocotyl tissue. While the enzyme could not be purified using any of these methods, it was possible to successfully conduct a more detailed biochemical analysis of the H + - ATPase. The molecular weight and isoelectric point of the enzyme were determined using N,N'dicyclohexylcarbodiimide (DCCD) and a H + - ATPase antibody. When plasma membrane vesicles were incubated with 20 μM [ 14 C]-DCCD at 0 C, a single 97,000 dalton protein was apparent on a fluorograph. A close correlation between [ 14 C]-DCCD labelling of the 97,000 dalton protein and the extent of ATPase inhibition over a range of DCCD concentrations suggests that this 97,000 dalton protein is a component of the plasma membrane H + - ATPase. An antibody raised against the plasma membrane H + - ATPase of Neurospora crassa cross-reacted with the 97,000 dalton DCCD-binding protein, further supporting the identity of this protein. Immunoblots of two dimensional gels of red beet plasma membrane vesicles indicated the isoelectric point of the enzyme to be pH 6.5

Na+,K+-ATPase concentration in rodent and human heart and skeletal muscle

DEFF Research Database (Denmark)

Kjeldsen, K; Bjerregaard, P; Richter, Erik

1988-01-01

rats, cardiomyopathic hamsters, and human subjects. These methods have earlier been shown to quantify the Na+,K+-ATPase concentration in muscle tissue with high accuracy. When rats were swim trained for six weeks the heart ventricular muscle Na+,K+-ATPase concentration was increased by 20% (p less than...... was increased by up to 46% (p less than 0.001) and decreased by up to 30% (p less than 0.005) after training and immobilisation respectively. Cardiomyopathic hamsters showed a reduction of 33% (p less than 0.005) in the heart ventricular Na+,K+-ATPase concentration compared with normal hamsters. This decrease...

Structural studies of P-type ATPase–ligand complexes using an X-ray free-electron laser

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Maike Bublitz

2015-07-01

Full Text Available Membrane proteins are key players in biological systems, mediating signalling events and the specific transport of e.g. ions and metabolites. Consequently, membrane proteins are targeted by a large number of currently approved drugs. Understanding their functions and molecular mechanisms is greatly dependent on structural information, not least on complexes with functionally or medically important ligands. Structure determination, however, is hampered by the difficulty of obtaining well diffracting, macroscopic crystals. Here, the feasibility of X-ray free-electron-laser-based serial femtosecond crystallography (SFX for the structure determination of membrane protein–ligand complexes using microcrystals of various native-source and recombinant P-type ATPase complexes is demonstrated. The data reveal the binding sites of a variety of ligands, including lipids and inhibitors such as the hallmark P-type ATPase inhibitor orthovanadate. By analyzing the resolution dependence of ligand densities and overall model qualities, SFX data quality metrics as well as suitable refinement procedures are discussed. Even at relatively low resolution and multiplicity, the identification of ligands can be demonstrated. This makes SFX a useful tool for ligand screening and thus for unravelling the molecular mechanisms of biologically active proteins.

Computer modelling reveals new conformers of the ATP binding loop of Na+/K+-ATPase involved in the transphosphorylation process of the sodium pump.

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Tejral, Gracian; Sopko, Bruno; Necas, Alois; Schoner, Wilhelm; Amler, Evzen

2017-01-01

Hydrolysis of ATP by Na + /K + -ATPase, a P-Type ATPase, catalyzing active Na + and K + transport through cellular membranes leads transiently to a phosphorylation of its catalytical α -subunit. Surprisingly, three-dimensional molecular structure analysis of P-type ATPases reveals that binding of ATP to the N-domain connected by a hinge to the P-domain is much too far away from the Asp 369 to allow the transfer of ATP's terminal phosphate to its aspartyl-phosphorylation site. In order to get information for how the transfer of the γ -phosphate group of ATP to the Asp 369 is achieved, analogous molecular modeling of the M 4 -M 5 loop of ATPase was performed using the crystal data of Na + /K + -ATPase of different species. Analogous molecular modeling of the cytoplasmic loop between Thr 338 and Ile 760 of the α 2 -subunit of Na + /K + -ATPase and the analysis of distances between the ATP binding site and phosphorylation site revealed the existence of two ATP binding sites in the open conformation; the first one close to Phe 475 in the N-domain, the other one close to Asp 369 in the P-domain. However, binding of Mg 2+ •ATP to any of these sites in the "open conformation" may not lead to phosphorylation of Asp 369 . Additional conformations of the cytoplasmic loop were found wobbling between "open conformation"  "semi-open conformation  "closed conformation" in the absence of 2Mg 2+ •ATP. The cytoplasmic loop's conformational change to the "semi-open conformation"-characterized by a hydrogen bond between Arg 543 and Asp 611 -triggers by binding of 2Mg 2+ •ATP to a single ATP site and conversion to the "closed conformation" the phosphorylation of Asp 369 in the P-domain, and hence the start of Na + /K + -activated ATP hydrolysis.

The modulation of erythrocyte Na+/K+-ATPase activity by curcumin

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Prabhakar Singh

2015-11-01

Full Text Available Curcumin, an active biphenolic molecule present in turmeric (Curcuma longa, has been reported to elicit plethora of health protective effects. The present study was carried out in vitro, in vivo and in silico to investigate the modulatory effects of curcumin on erythrocyte membrane Na+/K+-ATPase activity. In vitro curcumin (10−5 M to 10−8 M was incubated with human erythrocytes membrane. In vivo curcumin (340 mg/kg b.w. and 170 mg/kg b.w. was supplemented to wistar rats for 21 days. In silico, catalytic unit α of Na+/K+-ATPase (3b8e.pdb protein was used as a receptor for the natural ligand ATP to study curcumin-mediated docking simulation using AutoDock4. The in vitro effect of curcumin on the Na+/K+-ATPase activity in human erythrocytes was biphasic. An inhibitory response was observed at 10−5 M (p < 0.001. An activation of the Na+/K+-ATPase activity was observed at 10−7 and 10−8 M (p < 0.001 and p < 0.01. In vivo, curcumin supplementation to rats increased the Na+/K+-ATPase activity at doses 340 mg/kg b.w. (p < 0.001 as well as at 170 mg/kg b.w., (p < 0.01. AutoDock4 docking simulation study showed that both ligands curcumin and ATP actively interacted with amino acids Glu214, Ser215, Glu216, Thr371, Asn377, Arg378, Met379, Arg438, Val440, Ala444, Lys451 and Asp586 at the catalytic cavity of Na+/K+-ATPase. ATP had more H bonding and hydrophobic interaction with active site amino acid residues compared to curcumin. These finding may explain some of the health beneficial properties of curcumin associated with deregulated Na+/K+-ATPase activity or ions homeostasis.

Proteasome Inhibition Contributed to the Cytotoxicity of Arenobufagin after Its Binding with Na, K-ATPase in Human Cervical Carcinoma HeLa Cells.

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Qingxi Yue

Full Text Available Although the possibility of developing cardiac steroids/cardiac glycosides as novel cancer therapeutic agents has been recognized, the mechanism of their anticancer activity is still not clear enough. Toad venom extract containing bufadienolides, which belong to cardiac steroids, has actually long been used as traditional Chinese medicine in clinic for cancer therapy in China. The cytotoxicity of arenobufagin, a bufadienolide isolated from toad venom, on human cervical carcinoma HeLa cells was checked. And, the protein expression profile of control HeLa cells and HeLa cells treated with arenobufagin for 48 h was analyzed using two-dimensional electrophoresis, respectively. Differently expressed proteins in HeLa cells treated with arenobufagin were identified and the pathways related to these proteins were mapped from KEGG database. Computational molecular docking was performed to verify the binding of arenobufagin and Na, K-ATPase. The effects of arenobufagin on Na, K-ATPase activity and proteasome activity of HeLa cells were checked. The protein-protein interaction network between Na, K-ATPase and proteasome was constructed and the expression of possible intermediate proteins ataxin-1 and translationally-controlled tumor protein in HeLa cells treated with arenobufagin was then checked. Arenobufagin induced apoptosis and G2/M cell cycle arrest in HeLa cells. The cytotoxic effect of arenobufagin was associated with 25 differently expressed proteins including proteasome-related proteins, calcium ion binding-related proteins, oxidative stress-related proteins, metabolism-related enzymes and others. The results of computational molecular docking revealed that arenobufagin was bound in the cavity formed by the transmembrane alpha subunits of Na, K-ATPase, which blocked the pathway of extracellular Na+/K+ cation exchange and inhibited the function of ion exchange. Arenobufagin inhibited the activity of Na, K-ATPase and proteasome, decreased the

Regulation of renal function and structure by the signaling Na/K-ATPase.

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Xie, Jeffrey X; Li, Xin; Xie, Zijian

2013-12-01

The Na/K-ATPase as an essential ion pump was discovered more than 50 years ago (Skou (1989) Biochim. Biophys. Acta 1000, 439-446; Feraille and Doucet (2001) Physiol. Rev. 81, 345-418). The signaling function of Na/K-ATPase has been gradually appreciated over the last 20 years, first from the studies of regulatory effects of ouabain on cardiac cell growth. Several reviews on this topic have been written during the last few years (Schoner and Scheiner-Bobis (2007) Am. J. Physiol. Cell. Physiol. 293, C509-C536; Xie and Cai (2003) Mol. Interv. 3, 157 - 168; Bagrov et al. (2009) Pharmacol. Rev. 61, 9-38; Tian and Xie (2008) Physiology 23, 205-211; Fontana et al. (2013) FEBS J. 280, 5450-5455; Blanco and Wallace (2013) Am. J. Physiol. Renal Physiol. 305, F797-F812). This article will focus on the molecular mechanism of Na/K-ATPase-mediated signal transduction and its potential regulatory role in renal physiology and diseases. © 2013 International Union of Biochemistry and Molecular Biology.

Models for the a subunits of the Thermus thermophilus V/A-ATPase and Saccharomyces cerevisiae V-ATPase enzymes by cryo-EM and evolutionary covariance

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Schep, Daniel G.; Rubinstein, John L.

2016-01-01

Rotary ATPases couple ATP synthesis or hydrolysis to proton translocation across a membrane. However, understanding proton translocation has been hampered by a lack of structural information for the membrane-embedded a subunit. The V/A-ATPase from the eubacterium Thermus thermophilus is similar in structure to the eukaryotic V-ATPase but has a simpler subunit composition and functions in vivo to synthesize ATP rather than pump protons. We determined the T. thermophilus V/A-ATPase structure by cryo-EM at 6.4 Å resolution. Evolutionary covariance analysis allowed tracing of the a subunit sequence within the map, providing a complete model of the rotary ATPase. Comparing the membrane-embedded regions of the T. thermophilus V/A-ATPase and eukaryotic V-ATPase from Saccharomyces cerevisiae allowed identification of the α-helices that belong to the a subunit and revealed the existence of previously unknown subunits in the eukaryotic enzyme. Subsequent evolutionary covariance analysis enabled construction of a model of the a subunit in the S. cerevisae V-ATPase that explains numerous biochemical studies of that enzyme. Comparing the two a subunit structures determined here with a structure of the distantly related a subunit from the bovine F-type ATP synthase revealed a conserved pattern of residues, suggesting a common mechanism for proton transport in all rotary ATPases. PMID:26951669

Na+/K+-ATPase and vacuolar-type H+-ATPase in the gills of the aquatic air-breathing fish Trichogaster microlepis in response to salinity variation.

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Huang, Chun-Yen; Chao, Pei-Lin; Lin, Hui-Chen

2010-03-01

The aquatic air-breathing fish, Trichogaster microlepis, can be found in fresh water and estuaries. We further evaluated the changes in two important osmoregulatory enzymes, Na(+)/K(+)-ATPase (NKA) and vacuolar-type H(+)-ATPase (VHA), in the gills when fish were subjected to deionized water (DW), fresh water (FW), and salinated brackish water (salinity of 10 g/L). Fish were sampled only 4 days after experimental transfer. The mortality, plasma osmolality, and Na(+) concentration were higher in 10 g/L acclimated fish, while their muscle water content decreased with elevated external salinity. The highest NKA protein abundance was found in the fish gills in 10 g/L, and NKA activity was highest in the DW and 10 g/L acclimated fish. The VHA protein levels were highest in 10 g/L, and VHA activity was highest in the DW treatment. From immunohistochemical results, we found three different cell populations: (1) NKA-immunoreactive (NKA-IR) cells, (2) both NKA-IR and HA-IR cells, and (3) HA-IR cells. NKA-IR cells in the lamellar and interlamellar regions significantly increased in DW and 10 g/L treatments. Only HA-IR cells in the lamellar region were significantly increased in DW. In the interlamellar region, there was no difference in the number of HA-IR cells among the three treated. From these results, T. microlepis exhibited osmoregulatory ability in DW and 10 g/L treatments. The cell types involved in ionic regulation were also examined with immunofluorescence staining; three ionocyte types were found which were similar to the zebrafish model. Copyright 2009 Elsevier B.V. All rights reserved.

Altered Regulation of type 3 Na+/H+ exchanger, type 1 Na+/HCO3- cotransporter, and Na+,K+-ATPase in the Kidney of Rats with Experimental Rhabdomyolysis

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Ma, Seong Kwon; Bae, Eun Hui; Lee, JongUn; Kim, Sun Young; Kim, Sung Zoo; Choi, Ki Chul

2007-01-01

Metabolic acidosis was shown to correlate with deterioration of renal function in patients with rhabdomyolysis. The present study was aimed to investigate whether the changes of type 3 Na+/H+ exchanger (NHE3), type 1 Na+/HCO3- cotransporter (NBC1), and Na+,K+-ATPase α1 subunit may play a role in the pathogenesis of metabolic acidosis in glycerol-induced experimental rhabdomyolysis. Male Sprague-Dawley rats were deprived of fluid intake for 24 hours, and then were injected with 50% glycerol in normal saline (10 mL/kg, intramuscularly). At 24 hours after the glycerol injection, rats were sacrificed by decapitation. Control rats were injected with normal saline. The protein expression of NHE3, NBC1 and Na+,K+-ATPase α1 subunit was determined in the cortex of the kidney by immunoblotting and immunohistochemistry. Following the treatment of glycerol, creatinine clearance was significantly decreased, and high anion gap metabolic acidosis developed. In the experimental group, the expression of Na+,K+-ATPase α1 subunit was significantly decreased in the cortex of the kidney. On the contrary, the expression of NHE3 and NBC1 was significantly increased. Immunohistochemical analyses confirmed the immunoblotting data. In conclusion, the coordinate up-regulation of NHE3 and NBC1 may play an adaptive role against the metabolic acidosis in glycerol-induced rhabdomyolysis. PMID:24459502

Human and rodent muscle Na(+)-K(+)-ATPase in diabetes related to insulin, starvation, and training

DEFF Research Database (Denmark)

Schmidt, T A; Hasselbalch, S; Farrell, P A

1994-01-01

cerebral cortex Na(+)-K(+)-ATPase concentration as a result of diabetes, semistarvation, or insulin treatment. In human subjects, Na(+)-K(+)-ATPase concentration in vastus lateralis muscle biopsies was 17 and 22% greater (P dependent diabetes...... mellitus (n = 24) and insulin-dependent diabetes mellitus (n = 7) than in control subjects (n = 8). A positive linear correlation between muscle Na(+)-K(+)-ATPase and plasma insulin concentrations was observed (r = 0.50, P = 0.006; n = 29). Thus, insulin seems a regulator of muscle Na......(+)-K(+)-ATPase concentration, reduction of muscle Na(+)-K(+)-ATPase concentration with untreated diabetes bears similarities with undernourishment, and physical conditioning may ameliorate the muscle Na(+)-K(+)-ATPase concentration decrease induced by diabetes....

Mechanisms Involving Ang II and MAPK/ERK1/2 Signaling Pathways Underlie Cardiac and Renal Alterations during Chronic Undernutrition

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Pereira-Acácio, Amaury; Luzardo, Ricardo; Sampaio, Luzia S.; Luna-Leite, Marcia A.; Lara, Lucienne S.; Einicker-Lamas, Marcelo; Panizzutti, Rogério; Madeira, Caroline; Vieira-Filho, Leucio D.; Castro-Chaves, Carmen; Ribeiro, Valdilene S.; Paixão, Ana D. O.; Medei, Emiliano; Vieyra, Adalberto

2014-01-01

Background Several studies have correlated protein restriction associated with other nutritional deficiencies with the development of cardiovascular and renal diseases. The driving hypothesis for this study was that Ang II signaling pathways in the heart and kidney are affected by chronic protein, mineral and vitamin restriction. Methodology/Principal Findings Wistar rats aged 90 days were fed from weaning with either a control or a deficient diet that mimics those used in impoverished regions worldwide. Such restriction simultaneously increased ouabain-insensitive Na+-ATPase and decreased (Na++K+)ATPase activity in the same proportion in cardiomyocytes and proximal tubule cells. Type 1 angiotensin II receptor (AT1R) was downregulated by that restriction in both organs, whereas AT2R decreased only in the kidney. The PKC/PKA ratio increased in both tissues and returned to normal values in rats receiving Losartan daily from weaning. Inhibition of the MAPK pathway restored Na+-ATPase activity in both organs. The undernourished rats presented expanded plasma volume, increased heart rate, cardiac hypertrophy, and elevated systolic pressure, which also returned to control levels with Losartan. Such restriction led to electrical cardiac remodeling represented by prolonged ventricular repolarization parameters, induced triggered activity, early after-depolarization and delayed after-depolarization, which were also prevented by Losartan. Conclusion/Significance The mechanisms responsible for these alterations are underpinned by an imbalance in the PKC- and PKA-mediated pathways, with participation of angiotensin receptors and by activation of the MAPK/ERK1/2 pathway. These cellular and molecular alterations culminate in cardiac electric remodeling and in the onset of hypertension in adulthood. PMID:24983243

Cardiac Abnormalities in Youth with Obesity and Type 2 Diabetes.

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Bacha, Fida; Gidding, Samuel S

2016-07-01

Childhood obesity has been linked to cardiovascular disease (CVD) risk in adulthood. Of great concern is the expected increase in the population's CVD burden in relation to childhood obesity. This is compounded by the risk related to chronic hyperglycemia exposure in youth with type 2 diabetes. We herein provide an overview of the spectrum of early cardiovascular disease manifestation in youth with obesity and type 2 diabetes, in particular abnormalities in cardiac structure and function. Cardiac remodeling and adverse target organ damage is already evident in the pediatric age group in children with obesity and type 2 diabetes. This supports the importance of intensifying obesity prevention efforts and early intervention to treat comorbidities of obesity in the pediatric age group to prevent cardiac events in early adulthood.

Effect of triorganotin compounds on calcium transport mechanisms in rat cardiac sarcoplasmic reticulum

International Nuclear Information System (INIS)

Cameron, J.A.; Kodavanti, P.R.S.; Yallapragada, P.R.; Desaiah, D.

1990-01-01

Although organotin compounds, in general, are neurotoxicants, recent studies indicate that these tin compounds affect heme metabolism as well as cardiovascular system. Sarcoplasmic reticulum (SR) calcium pump together with phosphorylation of phospholamban has an important role in myocardial contraction and relaxation. Since organotin compounds interfere with cardiovascular system, we have studied the in vitro as well as in vivo effects of tributyltin bromide (TBT), triethyltin bromide (TET) and trimethyltin chloride (TMT) on cardiac SR Ca 2+ -pump activity, in order to know the relative potency of these tin compounds. SR was isolated from heart ventricles of male Sprague-Dawley rats and used for in vitro studies. For in vivo studies, rats were treated orally in corn oil for 6 days with different doses of TET (0.5, 1.0 and 1.5 mg/kg/d), TMT (0.75, 1.5 and 2.5 mg/kg/d) and TBT (0.75, 1.5 and 2.5 mg/kg/d). Rats were sacrificed 24 hr after the last dosage and cardiac SR was prepared. Cardiac SR Ca 2+ -ATPase and 45 Ca-uptake were measured. All the three tin compounds inhibited Ca 2+ -ATPase and 45 Ca-uptake in vitro in a concentration dependent manner. The order of potency for Ca 2+ -ATPase as determined IC 50 , is TBT (2 uM) > TET (63 uM) > TMT (280 uM). For 45 Ca-uptake, if followed the same order i.e., TBT (0.35 uM) > TET (10 uM) > TMT (440 uM). In agreement with in vitro results, both SR Ca 2+ -ATPase and 45 Ca-uptake were significantly inhibited in rats treated with these tin compounds. These studies indicate that triorganotin compounds affect Ca 2+ -pumping mechanisms and thereby alter cardiac contraction-relaxation process

Na+/Ca2+ exchange and Na+/K+-ATPase in the heart

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Shattock, Michael J; Ottolia, Michela; Bers, Donald M; Blaustein, Mordecai P; Boguslavskyi, Andrii; Bossuyt, Julie; Bridge, John H B; Chen-Izu, Ye; Clancy, Colleen E; Edwards, Andrew; Goldhaber, Joshua; Kaplan, Jack; Lingrel, Jerry B; Pavlovic, Davor; Philipson, Kenneth; Sipido, Karin R; Xie, Zi-Jian

2015-01-01

This paper is the third in a series of reviews published in this issue resulting from the University of California Davis Cardiovascular Symposium 2014: Systems approach to understanding cardiac excitation–contraction coupling and arrhythmias: Na+ channel and Na+ transport. The goal of the symposium was to bring together experts in the field to discuss points of consensus and controversy on the topic of sodium in the heart. The present review focuses on cardiac Na+/Ca2+ exchange (NCX) and Na+/K+-ATPase (NKA). While the relevance of Ca2+ homeostasis in cardiac function has been extensively investigated, the role of Na+ regulation in shaping heart function is often overlooked. Small changes in the cytoplasmic Na+ content have multiple effects on the heart by influencing intracellular Ca2+ and pH levels thereby modulating heart contractility. Therefore it is essential for heart cells to maintain Na+ homeostasis. Among the proteins that accomplish this task are the Na+/Ca2+ exchanger (NCX) and the Na+/K+ pump (NKA). By transporting three Na+ ions into the cytoplasm in exchange for one Ca2+ moved out, NCX is one of the main Na+ influx mechanisms in cardiomyocytes. Acting in the opposite direction, NKA moves Na+ ions from the cytoplasm to the extracellular space against their gradient by utilizing the energy released from ATP hydrolysis. A fine balance between these two processes controls the net amount of intracellular Na+ and aberrations in either of these two systems can have a large impact on cardiac contractility. Due to the relevant role of these two proteins in Na+ homeostasis, the emphasis of this review is on recent developments regarding the cardiac Na+/Ca2+ exchanger (NCX1) and Na+/K+ pump and the controversies that still persist in the field. PMID:25772291

Quantitative measurement of membrane Na{sup +}-K{sup +} ATPase activity using thallium-201: comparison with rubidium-86

Energy Technology Data Exchange (ETDEWEB)

Lee, Jae Tae; Shon, Sang Kyun; Lee, Kyu Bo [School of Medicine, Kyungpook National Univ., Taegu (Korea, Republic of); Lee, In Kyu [School of Medicine, Kyemyung Univ., Taegu (Korea, Republic of)

1998-06-01

Na{sup +}-K{sup +} ATPase activity has been estimated by the degree of inhibition of cation transport by cardiac glycosides (ouabain) using Rb-86 as a substrate. The biological characteristics of Tl-201 is known to be similar to those of potassium as a transport substrate in the presence of glucose, insulin or phobol myristate acetate (PMA). The purpose of this study was to measure ouabain sensitive Na{sup +}-K{sup +} ATPase activity using Tl-201 and compare with that using Rb-86. Smooth muscle cells isolated from rat aorta or human placental umbilical artery were cultured, and used to measure cellular Na{sup +}-K{sup +} ATPase activity. Na{sup +}-K{sup +} ATPase activity was measured as a percentage decrease in cellular uptake of Tl-201 or Rb-86 by ouabain under the presence of glucose, insulin or PMA in media. Na{sup +}-K{sup +} ATPase activity measured with Tl-201, as a transport substrate, was not different from those measured with Rb-86 in rat or human smooth muscle cell preparation. Incubation with high concentration glucose resulted in about 30% decrease in enzyme activity. In contrast, insulin or PMA resulted in 50-70% or 28% increase from baseline activity, respectively. These results suggests that Tl-201 could replace Rb-86 in measurement of ouabain sensitive Na{sup +}-K{sup +} ATPase activity in vitro. High level of glucose concentration decreased cellular Na{sup +}-K{sup +} ATPase activity, but insulin or PMA increased it.

Strong transthyretin immunostaining: potential pitfall in cardiac amyloid typing.

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Satoskar, Anjali A; Efebera, Yvonne; Hasan, Ayesha; Brodsky, Sergey; Nadasdy, Gyongyi; Dogan, Ahmet; Nadasdy, Tibor

2011-11-01

Although systemic amyloidosis commonly presents with renal disease, cardiac involvement usually determines the patient's prognosis. Cardiac involvement is seen in light chain amyloid and transthyretin amyloidosis. Distinguishing between these two is critical because prognosis and treatment differ. Our study demonstrates the unreliability of transthyretin immunostaining in subtyping cardiac amyloid. Between January 2003 and August 2010, we retrieved 229 native endomyocardial biopsies, of which 24 had amyloid. Immunohistochemistry for κ, λ, transthyretin, and serum amyloid A protein was performed on formalin-fixed, paraffin-embedded sections. Staining was graded as weak (trace to 1+) or strong (2 to 3+). Mass spectrometry (MS)-based proteomic typing of microdissected amyloid material was performed on selected cases. Fifteen patients had monoclonal gammopathy/plasma cell dyscrasia with cardiac amyloid. Eight of them (53%) showed strong transthyretin staining in the cardiac amyloid deposits. MS was performed in 5 of these 8 biopsies, and all 5 biopsies revealed light chain amyloid-type amyloid. Two of these 5 light chain amyloid biopsies did not even have concomitant strong staining for the appropriate light chain. Among the 15 cases with plasma cell dyscrasia, only 7 biopsies showed strong staining for the corresponding monoclonal light chain. Strong, false-positive immunostaining for transthyretin in cardiac amyloid is a potential pitfall, augmented by the frequent lack of staining for immunoglobulin light chains. Therefore, the presence of amyloid in the cardiac biopsy should prompt a search for plasma cell dyscrasia irrespective of transthyretin staining. Confirmation with MS should be sought, particularly if there is any discrepancy between κ/λ staining and serum immunofixation results.

Selective Assembly of Na,K-ATPase α2β2 Heterodimers in the Heart: DISTINCT FUNCTIONAL PROPERTIES AND ISOFORM-SELECTIVE INHIBITORS.

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Habeck, Michael; Tokhtaeva, Elmira; Nadav, Yotam; Ben Zeev, Efrat; Ferris, Sean P; Kaufman, Randal J; Bab-Dinitz, Elizabeta; Kaplan, Jack H; Dada, Laura A; Farfel, Zvi; Tal, Daniel M; Katz, Adriana; Sachs, George; Vagin, Olga; Karlish, Steven J D

2016-10-28

The Na,K-ATPase α 2 subunit plays a key role in cardiac muscle contraction by regulating intracellular Ca 2+ , whereas α 1 has a more conventional role of maintaining ion homeostasis. The β subunit differentially regulates maturation, trafficking, and activity of α-β heterodimers. It is not known whether the distinct role of α 2 in the heart is related to selective assembly with a particular one of the three β isoforms. We show here by immunofluorescence and co-immunoprecipitation that α 2 is preferentially expressed with β 2 in T-tubules of cardiac myocytes, forming α 2 β 2 heterodimers. We have expressed human α 1 β 1 , α 2 β 1 , α 2 β 2 , and α 2 β 3 in Pichia pastoris, purified the complexes, and compared their functional properties. α 2 β 2 and α 2 β 3 differ significantly from both α 2 β 1 and α 1 β 1 in having a higher K 0.5 K + and lower K 0.5 Na + for activating Na,K-ATPase. These features are the result of a large reduction in binding affinity for extracellular K + and shift of the E 1 P-E 2 P conformational equilibrium toward E 1 P. A screen of perhydro-1,4-oxazepine derivatives of digoxin identified several derivatives (e.g. cyclobutyl) with strongly increased selectivity for inhibition of α 2 β 2 and α 2 β 3 over α 1 β 1 (range 22-33-fold). Molecular modeling suggests a possible basis for isoform selectivity. The preferential assembly, specific T-tubular localization, and low K + affinity of α 2 β 2 could allow an acute response to raised ambient K + concentrations in physiological conditions and explain the importance of α 2 β 2 for cardiac muscle contractility. The high sensitivity of α 2 β 2 to digoxin derivatives explains beneficial effects of cardiac glycosides for treatment of heart failure and potential of α 2 β 2 -selective digoxin derivatives for reducing cardiotoxicity. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Lead reduces tension development and the myosin ATPase activity of the rat right ventricular myocardium

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D.V. Vassallo

2008-09-01

Full Text Available Lead (Pb2+ poisoning causes hypertension, but little is known regarding its acute effects on cardiac contractility. To evaluate these effects, force was measured in right ventricular strips that were contracting isometrically in 45 male Wistar rats (250-300 g before and after the addition of increasing concentrations of lead acetate (3, 7, 10, 30, 70, 100, and 300 µM to the bath. Changes in rate of stimulation (0.1-1.5 Hz, relative potentiation after pauses of 15, 30, and 60 s, effect of Ca2+ concentration (0.62, 1.25, and 2.5 mM, and the effect of isoproterenol (20 ng/mL were determined before and after the addition of 100 µM Pb2+. Effects on contractile proteins were evaluated after caffeine treatment using tetanic stimulation (10 Hz and measuring the activity of the myosin ATPase. Pb2+ produced concentration-dependent force reduction, significant at concentrations greater than 30 µM. The force developed in response to increasing rates of stimulation became smaller at 0.5 and 0.8 Hz. Relative potentiation increased after 100 µM Pb2+ treatment. Extracellular Ca2+ increment and isoproterenol administration increased force development but after 100 µM Pb2+ treatment the force was significantly reduced suggesting an effect of the metal on the sarcolemmal Ca2+ influx. Concentration of 100 µM Pb2+ also reduced the peak and plateau force of tetanic contractions and reduced the activity of the myosin ATPase. Results showed that acute Pb2+ administration, although not affecting the sarcoplasmic reticulum activity, produces a concentration-dependent negative inotropic effect and reduces myosin ATPase activity. Results suggest that acute lead administration reduced myocardial contractility by reducing sarcolemmal calcium influx and the myosin ATPase activity. These results also suggest that lead exposure is hazardous and has toxicological consequences affecting cardiac muscle.

Arginine substitution of a cysteine in transmembrane helix M8 converts Na+,K+-ATPase to an electroneutral pump similar to H+,K+-ATPase.

Science.gov (United States)

Holm, Rikke; Khandelwal, Jaanki; Einholm, Anja P; Andersen, Jens P; Artigas, Pablo; Vilsen, Bente

2017-01-10

Na + ,K + -ATPase and H + ,K + -ATPase are electrogenic and nonelectrogenic ion pumps, respectively. The underlying structural basis for this difference has not been established, and it has not been revealed how the H + ,K + -ATPase avoids binding of Na + at the site corresponding to the Na + -specific site of the Na + ,K + -ATPase (site III). In this study, we addressed these questions by using site-directed mutagenesis in combination with enzymatic, transport, and electrophysiological functional measurements. Replacement of the cysteine C932 in transmembrane helix M8 of Na + ,K + -ATPase with arginine, present in the H + ,K + -ATPase at the corresponding position, converted the normal 3Na + :2K + :1ATP stoichiometry of the Na + ,K + -ATPase to electroneutral 2Na + :2K + :1ATP stoichiometry similar to the electroneutral transport mode of the H + ,K + -ATPase. The electroneutral C932R mutant of the Na + ,K + -ATPase retained a wild-type-like enzyme turnover rate for ATP hydrolysis and rate of cellular K + uptake. Only a relatively minor reduction of apparent Na + affinity for activation of phosphorylation from ATP was observed for C932R, whereas replacement of C932 with leucine or phenylalanine, the latter of a size comparable to arginine, led to spectacular reductions of apparent Na + affinity without changing the electrogenicity. From these results, in combination with structural considerations, it appears that the guanidine + group of the M8 arginine replaces Na + at the third site, thus preventing Na + binding there, although allowing Na + to bind at the two other sites and become transported. Hence, in the H + ,K + -ATPase, the ability of the M8 arginine to donate an internal cation binding at the third site is decisive for the electroneutral transport mode of this pump.

Photosynthesis Activates Plasma Membrane H+-ATPase via Sugar Accumulation.

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Okumura, Masaki; Inoue, Shin-Ichiro; Kuwata, Keiko; Kinoshita, Toshinori

2016-05-01

Plant plasma membrane H(+)-ATPase acts as a primary transporter via proton pumping and regulates diverse physiological responses by controlling secondary solute transport, pH homeostasis, and membrane potential. Phosphorylation of the penultimate threonine and the subsequent binding of 14-3-3 proteins in the carboxyl terminus of the enzyme are required for H(+)-ATPase activation. We showed previously that photosynthesis induces phosphorylation of the penultimate threonine in the nonvascular bryophyte Marchantia polymorpha However, (1) whether this response is conserved in vascular plants and (2) the process by which photosynthesis regulates H(+)-ATPase phosphorylation at the plasma membrane remain unresolved issues. Here, we report that photosynthesis induced the phosphorylation and activation of H(+)-ATPase in Arabidopsis (Arabidopsis thaliana) leaves via sugar accumulation. Light reversibly phosphorylated leaf H(+)-ATPase, and this process was inhibited by pharmacological and genetic suppression of photosynthesis. Immunohistochemical and biochemical analyses indicated that light-induced phosphorylation of H(+)-ATPase occurred autonomously in mesophyll cells. We also show that the phosphorylation status of H(+)-ATPase and photosynthetic sugar accumulation in leaves were positively correlated and that sugar treatment promoted phosphorylation. Furthermore, light-induced phosphorylation of H(+)-ATPase was strongly suppressed in a double mutant defective in ADP-glucose pyrophosphorylase and triose phosphate/phosphate translocator (adg1-1 tpt-2); these mutations strongly inhibited endogenous sugar accumulation. Overall, we show that photosynthesis activated H(+)-ATPase via sugar production in the mesophyll cells of vascular plants. Our work provides new insight into signaling from chloroplasts to the plasma membrane ion transport mechanism. © 2016 American Society of Plant Biologists. All Rights Reserved.

Mutation of the Na+/K+-ATPase Atp1a1a.1 causes QT interval prolongation and bradycardia in zebrafish.

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Pott, Alexander; Bock, Sarah; Berger, Ina M; Frese, Karen; Dahme, Tillman; Keßler, Mirjam; Rinné, Susanne; Decher, Niels; Just, Steffen; Rottbauer, Wolfgang

2018-05-08

The genetic underpinnings that orchestrate the vertebrate heart rate are not fully understood yet, but of high clinical importance, since diseases of cardiac impulse formation and propagation are common and severe human arrhythmias. To identify novel regulators of the vertebrate heart rate, we deciphered the pathogenesis of the bradycardia in the homozygous zebrafish mutant hiphop (hip) and identified a missense-mutation (N851K) in Na + /K + -ATPase α1-subunit (atp1a1a.1). N851K affects zebrafish Na + /K + -ATPase ion transport capacity, as revealed by in vitro pump current measurements. Inhibition of the Na + /K + -ATPase in vivo indicates that hip rather acts as a hypomorph than being a null allele. Consequently, reduced Na + /K + -ATPase function leads to prolonged QT interval and refractoriness in the hip mutant heart, as shown by electrocardiogram and in vivo electrical stimulation experiments. We here demonstrate for the first time that Na + /K + -ATPase plays an essential role in heart rate regulation by prolonging myocardial repolarization. Copyright © 2018. Published by Elsevier Ltd.

Lithium-induced inhibition of Na-K ATPase and Ca ATPase activities in rat brain synaptosome.

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Cho, Y. W.

1995-01-01

To explore the action mechanism of lithium in the brain, the author investigated the effects of lithium on Na-K ATPase and Ca ATPase in rat brain synaptosomes prepared from forebrains by the method of Booth and Clark. The activities of Na-K ATPase and Ca ATPase were assayed by the level of inorganic phosphate liberated from the hydrolysis of ATP. Lithium at the optimum therapeutic concentration of 1 mM decreased the activity of Na-K ATPase from the control value of 19.08 +/- 0.29 to 18.27 +/- 0.10 micromoles Pi/mg protein/h and also reduced the activity of Ca ATPase from 6.38 +/- 0.12 to 5.64 +/- 0.12 micromoles Pi/mg protein/h. The decreased activity of Na-K ATPase will decrease the rate of Ca2+ efflux, probably via an Na-Ca exchange mechanism and will increase the rate of Ca2+ entry by the depolarization of nerve terminals. The reduced activity of Ca ATPase will result in the decreased efflux of Ca2+. As a Conclusion, it can be speculated that lithium elevates the intrasynaptosomal Ca2+ concentration via inhibition of the activities of Na-K ATPase and Ca ATPase, and this increased [Ca2+]i will cause the release of neurotransmitters and neurological effects of lithium. PMID:7598829

Computer modelling reveals new conformers of the ATP binding loop of Na+/K+-ATPase involved in the transphosphorylation process of the sodium pump

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Gracian Tejral

2017-03-01

Full Text Available Hydrolysis of ATP by Na+/K+-ATPase, a P-Type ATPase, catalyzing active Na+ and K+ transport through cellular membranes leads transiently to a phosphorylation of its catalytical α-subunit. Surprisingly, three-dimensional molecular structure analysis of P-type ATPases reveals that binding of ATP to the N-domain connected by a hinge to the P-domain is much too far away from the Asp369 to allow the transfer of ATP’s terminal phosphate to its aspartyl-phosphorylation site. In order to get information for how the transfer of the γ-phosphate group of ATP to the Asp369 is achieved, analogous molecular modeling of the M4–M5 loop of ATPase was performed using the crystal data of Na+/K+-ATPase of different species. Analogous molecular modeling of the cytoplasmic loop between Thr338 and Ile760 of the α2-subunit of Na+/K+-ATPase and the analysis of distances between the ATP binding site and phosphorylation site revealed the existence of two ATP binding sites in the open conformation; the first one close to Phe475 in the N-domain, the other one close to Asp369 in the P-domain. However, binding of Mg2+•ATP to any of these sites in the “open conformation” may not lead to phosphorylation of Asp369. Additional conformations of the cytoplasmic loop were found wobbling between “open conformation”  “semi-open conformation  “closed conformation” in the absence of 2Mg2+•ATP. The cytoplasmic loop’s conformational change to the “semi-open conformation”—characterized by a hydrogen bond between Arg543 and Asp611—triggers by binding of 2Mg2+•ATP to a single ATP site and conversion to the “closed conformation” the phosphorylation of Asp369 in the P-domain, and hence the start of Na+/K+-activated ATP hydrolysis.

Further investigations on the inorganic phosphate binding site of beef heart mitochondrial F1-ATPase

International Nuclear Information System (INIS)

Pougeois, R.; Lauquin, G.J.

1985-01-01

The possibility that 4-azido-2-nitrophenyl phosphate (ANPP), a photoreactive derivative of inorganic phosphate (P /sub i/ ), could mimic ATP was investigated. ANPP was hydrolyzed in the dark by sarcoplasmic reticulum Ca 2+ -ATPase in the presence of Ca 2+ but not in the presence of ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid. ANPP was not hydrolyzed by purified mitochondrial F1-ATPase; however, ADP and ATP protected F1-ATPase against ANPP photoinactivation. On the other hand, the trinitrophenyl nucleotide analogues (TNP-ADP, TNP-ATP, and TNP-AMP-PNP), which bind specifically at the two catalytic sites of F1-ATPase, abolished P /sub i/ binding on F1-ATPase; they do not protect F1-ATPase against ANPP photoinactivation. Furthermore, ANPP-photoinactivated F1-ATPase binds the TNP analogues in the same way as the native enzyme. The Pi binding site of F1-ATPase, which is shown to be photolabeled by ANPP, does not appear to be at the gamma-phosphate position of the catalytic sites

Regulation of alpha1 Na/K-ATPase expression by cholesterol.

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Chen, Yiliang; Li, Xin; Ye, Qiqi; Tian, Jiang; Jing, Runming; Xie, Zijian

2011-04-29

We have reported that α1 Na/K-ATPase regulates the trafficking of caveolin-1 and consequently alters cholesterol distribution in the plasma membrane. Here, we report the reciprocal regulation of α1 Na/K-ATPase by cholesterol. Acute exposure of LLC-PK1 cells to methyl β-cyclodextrin led to parallel decreases in cellular cholesterol and the expression of α1 Na/K-ATPase. Cholesterol repletion fully reversed the effect of methyl β-cyclodextrin. Moreover, inhibition of intracellular cholesterol trafficking to the plasma membrane by compound U18666A had the same effect on α1 Na/K-ATPase. Similarly, the expression of α1, but not α2 and α3, Na/K-ATPase was significantly reduced in the target organs of Niemann-Pick type C mice where the intracellular cholesterol trafficking is blocked. Mechanistically, decreases in the plasma membrane cholesterol activated Src kinase and stimulated the endocytosis and degradation of α1 Na/K-ATPase through Src- and ubiquitination-dependent pathways. Thus, the new findings, taken together with what we have already reported, revealed a previously unrecognized feed-forward mechanism by which cells can utilize the Src-dependent interplay among Na/K-ATPase, caveolin-1, and cholesterol to effectively alter the structure and function of the plasma membrane.

Protein phosphatase 2A interacts with the Na,K-ATPase and modulates its trafficking by inhibition of its association with arrestin.

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Toru Kimura

Full Text Available The P-type ATPase family constitutes a collection of ion pumps that form phosphorylated intermediates during ion transport. One of the best known members of this family is the Na⁺,K⁺-ATPase. The catalytic subunit of the Na⁺,K⁺-ATPase includes several functional domains that determine its enzymatic and trafficking properties.Using the yeast two-hybrid system we found that protein phosphatase 2A (PP2A catalytic C-subunit is a specific Na⁺,K⁺-ATPase interacting protein. PP-2A C-subunit interacted with the Na⁺,K⁺-ATPase, but not with the homologous sequences of the H⁺,K⁺-ATPase. We confirmed that the Na⁺,K⁺-ATPase interacts with a complex of A- and C-subunits in native rat kidney. Arrestins and G-protein coupled receptor kinases (GRKs are important regulators of G-protein coupled receptor (GPCR signaling, and they also regulate Na⁺,K⁺-ATPase trafficking through direct association. PP2A inhibits association between the Na⁺,K⁺-ATPase and arrestin, and diminishes the effect of arrestin on Na⁺,K⁺-ATPase trafficking. GRK phosphorylates the Na⁺,K⁺-ATPase and PP2A can at least partially reverse this phosphorylation.Taken together, these data demonstrate that the sodium pump belongs to a growing list of ion transport proteins that are regulated through direct interactions with the catalytic subunit of a protein phosphatase.

Membrane-bound ATPase contributes to hop resistance of Lactobacillus brevis

NARCIS (Netherlands)

Sakamoto, K; van Veen, HW; Saito, H; Kobayashi, H; Konings, WN

2002-01-01

The activity of the membrane-bound H+-ATPase of the beer spoilage bacterium Lactobacillus brevis ABBC45 increased upon adaptation to bacteriostatic hop compounds. The ATPase activity was optimal around pH 5.6 and increased up to fourfold when L. brevis was exposed to 666 muM hop compounds. The

Cardiac parasympathetic outflow during dynamic exercise in humans estimated from power spectral analysis of P-P interval variability.

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Takahashi, Makoto; Nakamoto, Tomoko; Matsukawa, Kanji; Ishii, Kei; Watanabe, Tae; Sekikawa, Kiyokazu; Hamada, Hironobu

2016-03-01

What is the central question of this study? Should we use the high-frequency (HF) component of P-P interval as an index of cardiac parasympathetic nerve activity during moderate exercise? What is the main finding and its importance? The HF component of P-P interval variability remained even at a heart rate of 120-140 beats min(-1) and was further reduced by atropine, indicating incomplete cardiac vagal withdrawal during moderate exercise. The HF component of R-R interval is invalid as an estimate of cardiac parasympathetic outflow during moderate exercise; instead, the HF component of P-P interval variability should be used. The high-frequency (HF) component of R-R interval variability has been widely used as an indirect estimate of cardiac parasympathetic (vagal) outflow to the sino-atrial node of the heart. However, we have recently found that the variability of the R-R interval becomes much smaller during dynamic exercise than that of the P-P interval above a heart rate (HR) of ∼100 beats min(-1). We hypothesized that cardiac parasympathetic outflow during dynamic exercise with a higher intensity may be better estimated using the HF component of P-P interval variability. To test this hypothesis, the HF components of both P-P and R-R interval variability were analysed using a Wavelet transform during dynamic exercise. Twelve subjects performed ergometer exercise to increase HR from the baseline of 69 ± 3 beats min(-1) to three different levels of 100, 120 and 140 beats min(-1). We also examined the effect of atropine sulfate on the HF components in eight of the 12 subjects during exercise at an HR of 140 beats min(-1) . The HF component of P-P interval variability was significantly greater than that of R-R interval variability during exercise, especially at the HRs of 120 and 140 beats min(-1). The HF component of P-P interval variability was more reduced by atropine than that of R-R interval variability. We conclude that cardiac parasympathetic outflow to the

Modification of the Neurospora crassa plasma membrane [H+]-ATPase with N,N'-dicyclohexycarbodiimide

International Nuclear Information System (INIS)

Sussman, M.R.; Slayman, C.W.

1983-01-01

The carboxyl-modifying reagent N,N'-dicyclohexylcarbodiimide (DCCD) inactivates the ATPase with pseudo-first order kinetics, suggesting that one site on the enzyme is involved. The rate constant for inactivation at pH 7.5 and 30 0 C is approximately 1000 M -1 min -1 , similar to values reported for the DCCD-binding proteolipid of F 0 -F 1 -type [H + ]-ATPases and for the sarcoplasmic reticulum [Ca +2 ]-ATPase. Although hydrophobic carbodiimides are inhibitory at micromolar concentrations, a hydrophilic analogue, 1-ethyl-3-(dimethylaminopropyl)-carbodiimide, is completely inactive even at millimolar concentrations. This result implies that the DCCD-reactive site is located in a lipophilic environment. [ 14 C]DCCD is incorporated into the M/sub r/ = 104,000 polypeptide at a rate similar to the rate of inactivation. There is no evidence for a separate low molecular weight DCCD-binding proteolipid. Using quantitative amino acid analysis, we established that complete inhibition occurs at a stoichiometry of 0.4 mol of DCCD/mol of polypeptide. Overall, the results are consistent with the idea the DCCD reacts with a single amino acid residue of the Neuspora [H + ]-ATPase, thereby blcoking ATP hydrolysis and proton translocation. 21 references, 5 figures, 2 tables

Purification, crystallization and preliminary X-ray crystallographic studies of the Mycobacterium tuberculosis DNA gyrase ATPase domain

International Nuclear Information System (INIS)

Roué, Mélanie; Agrawal, Alka; Volker, Craig; Mossakowska, Danuta; Mayer, Claudine; Bax, Benjamin D.

2013-01-01

The ATPase domain of M. tuberculosis DNA gyrase was crystallized using hanging-drop vapour diffusion. The crystals belonged to space groups P1 and P2 1 . Diffraction data were collected to resolutions of 2.9 and 3.3 Å, respectively. Mycobacterium tuberculosis DNA gyrase, a nanomachine involved in the regulation of DNA topology, is the only type II topoisomerase present in this organism and hence is the sole target of fluoroquinolones in the treatment of tuberculosis. The ATPase domain provides the energy required for catalysis by ATP hydrolysis. Two constructs corresponding to this 43 kDa domain, Mtb-GyrB47 C1 and Mtb-GyrB47 C2 , have been overproduced, purified and crystallized. Diffraction data were collected from three crystal forms. The crystals belonged to space groups P1 and P2 1 and diffracted to resolutions of 2.9 and 3.3 Å, respectively

Subcellular localization of H(+)-ATPase from pumpkin hypocotyls (Cucurbita maxima L.) by membrane fractionation.

Science.gov (United States)

Scherer, G F

1984-03-01

A new method of preparing sealed vesicles from membrane fractions of pumpkin hypocotyls in ethanolamine-containing buffers was used to investigate the subcellular localization of H(+)-ATPase measured as nigericin-stimulated ATPase. In a fluorescence-quench assay, the H(+) pump was directly demonstrated. The H(+) pump was substrate-specific for Mg·ATP and 0.1 mM diethylstilbestrol completely prevented the development of a Δ pH. The presence of unsupecific phosphatase hampered the detection of nigericin-stimulated ATPase. Unspecific phosphatases could be demonstrated by comparing the broad substrate specificity of the hydrolytic activities of the fractions with the clear preference for Mg·ATP as the substrate for the proton pump. Inhibitor studies showed that neither orthovanadate nor molybdate are absolutely specific for ATPase or acid phosphatase, respectively. Diethylstilbestrol seemed to be a specific inhibitor of ATPase activity in fractions containing nigericin-stimulated ATPase, but it stimulated acid phosphatase which tended to obscure its effect on ATPase activity. Nigericin-stimulated ATPase had its optimum at pH 6.0 and the nigericin effect was K(+)-dependent. The combination of valinomycin and carbonylcyanide m-chlorophenylhydrazone had a similar effect to nigericin, but singly these ionophores were much less stimulatory. After prolonged centrifugation on linear sucrose gradients, nigericin-stimulated ATPase correlated in dense fractions with plasma membrane markers but a part of it remained at the interphase. This lessdense part of the nigericin-stimulated ATPase could be derived from tonoplast vesicles because α-mannosidase, an enzyme of the vacuolar sap, remained in the upper part of the gradient. Nigericinstimulated ATPase did not correlate with the mitochondrial marker, cytochrome c oxidase, whereas azide inhibition of ATPase activity did.

Na,K-ATPase biostimulation by low-energy laser irradiation: comparative effects in membrane, solubilized and proteoliposomes enzyme

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Rigos, C.F.; Tedesco, A.C.; Ciancaglini, P. [Universidade de Sao Paulo (USP), Ribeirao Preto, SP (Brazil). Faculdade de Filosofia, Ciencias e Letras. Dept. de Quimica; Santos, H.L. [Universidade Federal de Sao Joao Del Rei (UFSJ), MG (Brazil)

2008-07-01

Full text: The mechanism of laser irradiation action on living cells is not yet understood. The role of membrane ATPases as possible targets has been analyzed. In our group we have been working with Na,K-ATPase. This enzyme is a member of the P-type family of active cation transport proteins. Thus, the aim of the present work was to investigate the effect of low-energy laser irradiation (685 nm, 35 mW) on the ATPase activity of different forms of the Na,K-ATPase. Membrane-bound and solubilized (ab)2 form of Na,K-ATPase was obtained from the rabbit kidney and DPPC:DPPE-proteoliposomes were prepared by the co-solubilization method. Irradiations were carried out at 685 nm. The ATPase activity of the membrane fraction was not altered with exposition to irradiation doses between 4 and 24 J/c m2. With irradiation doses ranging from 32 to 40 J/c m2, a 28% increase on the ATPase activity was observed while when using up to 50 J/c m2 no additional enhancement was observed. When bio stimulation was done using the purified or the reconstituted enzyme, an increase of about 36-40% on the ATPase activity was observed using only 4-8 J/c m2. With irradiation above these values (24 J/c m2) no additional increase in the activity appeared. These studies revealed that the bio stimulation of ATPase activity from different forms of the Na,K -ATPase is dose dependent in different ranges of irradiation exposure. The stimulation promoted by visible laser doses was modulated and the process was reverted after 2 h for the enzyme present in the membrane and after about 5 h for the solubilized or the reconstituted in DPPC:DPPE-liposomes.

Na,K-ATPase biostimulation by low-energy laser irradiation: comparative effects in membrane, solubilized and proteoliposomes enzyme

International Nuclear Information System (INIS)

Rigos, C.F.; Tedesco, A.C.; Ciancaglini, P.

2008-01-01

Full text: The mechanism of laser irradiation action on living cells is not yet understood. The role of membrane ATPases as possible targets has been analyzed. In our group we have been working with Na,K-ATPase. This enzyme is a member of the P-type family of active cation transport proteins. Thus, the aim of the present work was to investigate the effect of low-energy laser irradiation (685 nm, 35 mW) on the ATPase activity of different forms of the Na,K-ATPase. Membrane-bound and solubilized (ab)2 form of Na,K-ATPase was obtained from the rabbit kidney and DPPC:DPPE-proteoliposomes were prepared by the co-solubilization method. Irradiations were carried out at 685 nm. The ATPase activity of the membrane fraction was not altered with exposition to irradiation doses between 4 and 24 J/c m2. With irradiation doses ranging from 32 to 40 J/c m2, a 28% increase on the ATPase activity was observed while when using up to 50 J/c m2 no additional enhancement was observed. When bio stimulation was done using the purified or the reconstituted enzyme, an increase of about 36-40% on the ATPase activity was observed using only 4-8 J/c m2. With irradiation above these values (24 J/c m2) no additional increase in the activity appeared. These studies revealed that the bio stimulation of ATPase activity from different forms of the Na,K -ATPase is dose dependent in different ranges of irradiation exposure. The stimulation promoted by visible laser doses was modulated and the process was reverted after 2 h for the enzyme present in the membrane and after about 5 h for the solubilized or the reconstituted in DPPC:DPPE-liposomes

MicroRNAs and cardiac sarcoplasmic reticulum calcium ATPase-2 in human myocardial infarction: expression and bioinformatic analysis.

Science.gov (United States)

Boštjančič, Emanuela; Zidar, Nina; Glavač, Damjan

2012-10-15

Cardiac sarco(endo)plasmic reticulum calcium ATPase-2 (SERCA2) plays one of the central roles in myocardial contractility. Both, SERCA2 mRNA and protein are reduced in myocardial infarction (MI), but the correlation has not been always observed. MicroRNAs (miRNAs) act by targeting 3'-UTR mRNA, causing translational repression in physiological and pathological conditions, including cardiovascular diseases. One of the aims of our study was to identify miRNAs that could influence SERCA2 expression in human MI. The protein SERCA2 was decreased and 43 miRNAs were deregulated in infarcted myocardium compared to corresponding remote myocardium, analyzed by western blot and microRNA microarrays, respectively. All the samples were stored as FFPE tissue and in RNAlater. miRNAs binding prediction to SERCA2 including four prediction algorithms (TargetScan, PicTar, miRanda and mirTarget2) identified 213 putative miRNAs. TAM and miRNApath annotation of deregulated miRNAs identified 18 functional and 21 diseased states related to heart diseases, and association of the half of the deregulated miRNAs to SERCA2. Free-energy of binding and flanking regions (RNA22, RNAfold) was calculated for 10 up-regulated miRNAs from microarray analysis (miR-122, miR-320a/b/c/d, miR-574-3p/-5p, miR-199a, miR-140, and miR-483), and nine miRNAs deregulated from microarray analysis were used for validation with qPCR (miR-21, miR-122, miR-126, miR-1, miR-133, miR-125a/b, and miR-98). Based on qPCR results, the comparison between FFPE and RNAlater stored tissue samples, between Sybr Green and TaqMan approaches, as well as between different reference genes were also performed. Combing all the results, we identified certain miRNAs as potential regulators of SERCA2; however, further functional studies are needed for verification. Using qPCR, we confirmed deregulation of nine miRNAs in human MI, and show that qPCR normalization strategy is important for the outcome of miRNA expression analysis in human MI.

Muscle fibre type composition of a number of limb muscles in different types of horse.

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Snow, D H; Guy, P S

1980-03-01

Skeletal muscle of the equine was differentiated into three fibre types according to myosin ATPase (pH 9.4) and succinic dehydrogenase activity. The percentage of these types was determined in the musculus deltoideus, m triceps brachii caput longum, m gluteus medius, m semitendinosis, m biceps femoris and m vastus lateralis of the thoroughbred, Shetland pony, pony, heavy hunter and donkey. In addition the m gluteus medius was examined in the arab and American racing quarterhorse. High myosin ATPase activity fibres varied from a mean of 93.2 per cent in the m gluteus medius of the quarterhorse to 58.2 per cent in the m vastus lateralis of the donkey. In the m gluteus medius it was found that the percentage of high mycosin ATPase (pH 9.4) fibres varied significantly among breeds and these differences were related to the sprinting speed of the breed.

Increased anxiety and depression in Danish cardiac patients with a type D personality: cross-validation of the Type D Scale (DS14)

DEFF Research Database (Denmark)

Spindler, Helle; Kruse, Charlotte; Zwisler, Ann-Dorthe

2009-01-01

BACKGROUND: Type D personality is an emerging risk factor in cardiovascular disease. We examined the psychometric properties of the Danish version of the Type D Scale (DS14) and the impact of Type D on anxiety and depression in cardiac patients. METHOD: Cardiac patients (n = 707) completed the DS14......, the Hospital Anxiety and Depression Scale, and the Eysenck Personality Questionnaire. A subgroup (n = 318) also completed the DS14 at 3 or 12 weeks. RESULTS: The two-factor structure of the DS14 was confirmed; the subscales negative affectivity and social inhibition were shown to be valid, internally...... consistent (Cronbach's alpha = 0.87/0.91; mean inter-item correlations = 0.49/0.59), and stable over 3 and 12 weeks (r = 0.85/0.78; 0.83/0.79; ps depression (beta, 0.47; p

Bone Morphogenetic Protein 9 Reduces Cardiac Fibrosis and Improves Cardiac Function in Heart Failure.

Science.gov (United States)

Morine, Kevin J; Qiao, Xiaoying; York, Sam; Natov, Peter S; Paruchuri, Vikram; Zhang, Yali; Aronovitz, Mark J; Karas, Richard H; Kapur, Navin K

2018-02-27

Background -Heart failure is a growing cause of morbidity and mortality worldwide. Transforming growth factor beta (TGF-β1) promotes cardiac fibrosis, but also activates counter-regulatory pathways that serve to regulate TGF-β1 activity in heart failure. Bone morphogenetic protein 9 (BMP9) is a member of the TGFβ family of cytokines and signals via the downstream effector protein Smad1. Endoglin is a TGFβ co-receptor that promotes TGF-β1 signaling via Smad3 and binds BMP9 with high affinity. We hypothesized that BMP9 limits cardiac fibrosis by activating Smad1 and attenuating Smad3 and further that neutralizing endoglin activity promotes BMP9 activity. Methods -We examined BMP9 expression and signaling in human cardiac fibroblasts and human subjects with heart failure. We utilized the thoracic aortic constriction (TAC) induced model of heart failure to evaluate the functional effect of BMP9 signaling on cardiac remodeling. Results -BMP9 expression is increased in the circulation and left ventricle (LV) of human subjects with heart failure and is expressed by cardiac fibroblasts. Next, we observed that BMP9 attenuates Type I collagen synthesis in human cardiac fibroblasts using recombinant human BMP9 and an siRNA approach. In BMP9 -/- mice subjected to TAC, loss of BMP9 activity promotes cardiac fibrosis, impairs LV function, and increases LV levels of phosphorylated Smad3 (pSmad3), not pSmad1. In contrast, treatment of wild-type mice subjected to TAC with recombinant BMP9 limits progression of cardiac fibrosis, improves LV function, enhances myocardial capillary density, and increases LV levels of pSmad1, not pSmad3 compared to vehicle treated controls. Since endoglin binds BMP9 with high affinity, we explored the effect of reduced endoglin activity on BMP9 activity. Neutralizing endoglin activity in human cardiac fibroblasts or in wild-type mice subjected to TAC induced heart failure limits collagen production, increases BMP9 protein levels, and increases

Activity determination of Na+ K+ - ATPase and Mg++ - ATPase enzymes in the gill of Poecilia vivipara (Osteichthyes, Cyprinodontiformes in different salinities

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Marcelo da Cunha Amaral

2001-03-01

Full Text Available This work aimed to know the tolerance mechanisms through the salinity variation by Na+ K+ - ATPase and Mg++ - ATPase and enzymes encountered in the gills of Poecilia vivipara. In field, the presence of this species was observed in salinities of 0 and 28‰. In laboratory, these fish were maintained in aquarium with mean salinity of 30‰ and positive responses were obtained. Some adult specimens, collected in a lagoon of the Coqueiros Beach, were utilized as matrixes. In the experiments the specimens used were those born in the test aquarium. For each salinity studied three replicates were made with three specimens for each one. The alevins were maintained in salinities of 5, 10, 15, 20, 25, 30 and 35‰ during a month for adaptation. Gills were extracted in appropriate buffer for isolation of plasma membrane and used for specific dosage of the total enzymatic activity of Na+ K+ - ATPase and Mg++ - ATPase. The relation of alevins to their adaptation towards the salinity variation was also studied. The activity of the two enzymes showed a different result. The major expression of Na+ K+ - ATPase was observed in 20‰ (35 µmoles Pi.mg protein.h-1, the best salinity to cultivate P. vivipara.Este trabalho teve como objetivo conhecer os mecanismos de tolerância às variações de salinidade, pelas enzimas Mg++ - ATPase e Na+ K+ - ATPase, encontrada nas brânquias de Poecilia vivipara. No campo, foi observada a presença desta espécie em salinidades entre 0 e 28‰. No laboratório, os indivíduos foram mantidos em salinidade de 30‰ e responderam positivamente. Os indivíduos adultos, coletados em uma lagoa na praia dos Coqueiros, foram utilizados como matrizes. Nos experimentos foram usados alevinos que nasceram nos aquários testes. Para cada salinidade estudada foram feitas três réplicas com três espécimens em cada uma. Os alevinos foram mantidos em salinidades de 5, 10, 15, 20, 25, 30 e 35‰, durante um mês para total adaptação. As br

Influence of nitric acid synthesis on the function of (Na, K)-ATPase in the heart

International Nuclear Information System (INIS)

Vrbjar, N.; Bernathova, I.; Pechanova, O.

1998-01-01

Function of the sodium pump was characterized by kinetic parameters of the (Na,K)-ATPase at normal and lower synthesis of nitric oxide (NO) in the rat heart. Our findings indicate that there is no change in energy utilization by the cardiac sodium pump during lowered NO-synthesis. The transport properties of the enzyme are deteriorated, due to its decreased sensitivity to Na + . Inhibition of NO-synthesis in acute experiment by high doses of L-arginine analogue decreased the activity of (Na,K)-ATPase [Biol. Neonate, 68, 419 (1995)], an enzyme involved in the active translocation of Na + and K + ions across cell membranes. The present study was designed to investigate the influence of chronic inhibition of NO-synthesis on function of Na,K)-ATPase. One group (n=8) of adult male Wistar rats served as controls and the second group (n=16+ was treated with L-arginine analogue, the N G -nitro-L-arginine methyl ester (L-NAME) in a dose 40 mg/kg/day in drinking water for 4 weeks. NO-synthase activity was determined in crude homogenates of cardiac tissue by measuring the production of [ 3 H]-L-citrulline from from [ 3 H]-L-arginine. Chronic administration of L-NAME induced a significant inhibition (to 30%) of the NO-synthase activity. In control group the activity of NO-synthase amounted 11.44 ± 1.03 and in the L-NAME group it was 3.19 ± pmol L-Cit/min/mg protein. At the activation with ATP the shortage in NO-synthesis induced by L-NAME did not provoke significant changes in both, the V max and the K m value. This indicates that during the lower synthesis of NO there is no change in energy consumption for the transport of Na + and K + ions by the (Na,K)-ATPase. The stable value of K m indicates that shortage in NO does not induce changes in the vicinity of the ATP-binding site. On the other hand our results indicate that at lowered NO-synthesis the (Na,K)-ATPase in myocardium changes its Na + -binding and probably also the Na + -transport properties as it revealed from the

AAA-ATPases in Protein Degradation

Directory of Open Access Journals (Sweden)

Ravikiran S. Yedidi

2017-06-01

Full Text Available Proteolytic machineries containing multisubunit protease complexes and AAA-ATPases play a key role in protein quality control and the regulation of protein homeostasis. In these protein degradation machineries, the proteolytically active sites are formed by either threonines or serines which are buried inside interior cavities of cylinder-shaped complexes. In eukaryotic cells, the proteasome is the most prominent protease complex harboring AAA-ATPases. To degrade protein substrates, the gates of the axial entry ports of the protease need to be open. Gate opening is accomplished by AAA-ATPases, which form a hexameric ring flanking the entry ports of the protease. Protein substrates with unstructured domains can loop into the entry ports without the assistance of AAA-ATPases. However, folded proteins require the action of AAA-ATPases to unveil an unstructured terminus or domain. Cycles of ATP binding/hydrolysis fuel the unfolding of protein substrates which are gripped by loops lining up the central pore of the AAA-ATPase ring. The AAA-ATPases pull on the unfolded polypeptide chain for translocation into the proteolytic cavity of the protease. Conformational changes within the AAA-ATPase ring and the adjacent protease chamber create a peristaltic movement for substrate degradation. The review focuses on new technologies toward the understanding of the function and structure of AAA-ATPases to achieve substrate recognition, unfolding and translocation into proteasomes in yeast and mammalian cells and into proteasome-equivalent proteases in bacteria and archaea.

AAA-ATPases in Protein Degradation.

Science.gov (United States)

Yedidi, Ravikiran S; Wendler, Petra; Enenkel, Cordula

2017-01-01

Proteolytic machineries containing multisubunit protease complexes and AAA-ATPases play a key role in protein quality control and the regulation of protein homeostasis. In these protein degradation machineries, the proteolytically active sites are formed by either threonines or serines which are buried inside interior cavities of cylinder-shaped complexes. In eukaryotic cells, the proteasome is the most prominent protease complex harboring AAA-ATPases. To degrade protein substrates, the gates of the axial entry ports of the protease need to be open. Gate opening is accomplished by AAA-ATPases, which form a hexameric ring flanking the entry ports of the protease. Protein substrates with unstructured domains can loop into the entry ports without the assistance of AAA-ATPases. However, folded proteins require the action of AAA-ATPases to unveil an unstructured terminus or domain. Cycles of ATP binding/hydrolysis fuel the unfolding of protein substrates which are gripped by loops lining up the central pore of the AAA-ATPase ring. The AAA-ATPases pull on the unfolded polypeptide chain for translocation into the proteolytic cavity of the protease. Conformational changes within the AAA-ATPase ring and the adjacent protease chamber create a peristaltic movement for substrate degradation. The review focuses on new technologies toward the understanding of the function and structure of AAA-ATPases to achieve substrate recognition, unfolding and translocation into proteasomes in yeast and mammalian cells and into proteasome-equivalent proteases in bacteria and archaea.

123I-MIBG imaging detects cardiac involvement and predicts cardiac events in Churg-Strauss syndrome

International Nuclear Information System (INIS)

Horiguchi, Yoriko; Morita, Yukiko; Tsurikisawa, Naomi; Akiyama, Kazuo

2011-01-01

In Churg-Strauss syndrome (CSS) it is important to detect cardiac involvement, which predicts poor prognosis. This study evaluated whether 123 I-metaiodobenzylguanidine (MIBG) scintigraphy could detect cardiac damage and predict cardiac events in CSS. 123 I-MIBG scintigraphy was performed in 28 patients with CSS, 12 of whom had cardiac involvement. The early and delayed heart to mediastinum ratio (early H/M and delayed H/M) and washout rate were calculated by using 123 I-MIBG scintigraphy and compared with those in control subjects. Early H/M and delayed H/M were significantly lower and the washout rate was significantly higher in patients with cardiac involvement than in those without and in controls (early H/M, p = 0.0024, p = 0.0001; delayed H/M, p = 0.0002, p = 0.0001; washout rate, p = 0.0012, p = 0.0052 vs those without and vs controls, respectively). Accuracy for detecting cardiac involvement was 86% for delayed H/M and washout rate and 79% for early H/M and B-type natriuretic peptide (BNP). Kaplan-Meier analysis showed significantly lower cardiac event-free rates in patients with early H/M ≤ 2.18 and BNP > 21.8 pg/ml than those with early H/M > 2.18 and BNP ≤ 21.8 pg/ml (log-rank test p = 0.006). Cardiac sympathetic nerve function was damaged in CSS patients with cardiac involvement. 123 I-MIBG scintigraphy was useful in detecting cardiac involvement and in predicting cardiac events. (orig.)

Type D patients report poorer health status prior to and after cardiac rehabilitation compared to non-type D patients

DEFF Research Database (Denmark)

Pelle, Aline J; Erdman, Ruud A M; van Domburg, Ron T

2008-01-01

Type D personality is an emerging risk factor in coronary artery disease (CAD). Cardiac rehabilitation (CR) improves outcomes, but little is known about the effects of CR on Type D patients.......Type D personality is an emerging risk factor in coronary artery disease (CAD). Cardiac rehabilitation (CR) improves outcomes, but little is known about the effects of CR on Type D patients....

Response of plasma membrane H+-ATPase in rice (Oryza sativa) seedlings to simulated acid rain.

Science.gov (United States)

Liang, Chanjuan; Ge, Yuqing; Su, Lei; Bu, Jinjin

2015-01-01

Understanding the adaptation of plants to acid rain is important to find feasible approaches to alleviate such damage to plants. We studied effects of acid rain on plasma membrane H(+)-ATPase activity and transcription, intracellular H(+), membrane permeability, photosynthetic efficiency, and relative growth rate during stress and recovery periods. Simulated acid rain at pH 5.5 did not affect plasma membrane H(+)-ATPase activity, intracellular H(+), membrane permeability, photosynthetic efficiency, and relative growth rate. Plasma membrane H(+)-ATPase activity and transcription in leaves treated with acid rain at pH 3.5 was increased to maintain ion homeostasis by transporting excessive H(+) out of cells. Then intracellular H(+) was close to the control after a 5-day recovery, alleviating damage on membrane and sustaining photosynthetic efficiency and growth. Simulated acid rain at pH 2.5 inhibited plasma membrane H(+)-ATPase activity by decreasing the expression of H(+)-ATPase at transcription level, resulting in membrane damage and abnormal intracellular H(+), and reduction in photosynthetic efficiency and relative growth rate. After a 5-day recovery, all parameters in leaves treated with pH 2.5 acid rain show alleviated damage, implying that the increased plasma membrane H(+)-ATPase activity and its high expression were involved in repairing process in acid rain-stressed plants. Our study suggests that plasma membrane H(+)-ATPase can play a role in adaptation to acid rain for rice seedlings.

Visualizing the mapped ion pathway through the Na,K-ATPase pump.

Science.gov (United States)

Takeuchi, Ayako; Reyes, Nicolás; Artigas, Pablo; Gadsby, David C

2009-11-01

translocation by the Na(+),K(+)-ATPase and by other P-type pumps like the Ca(2+)- and H(+),K(+)-ATPases.

Visualizing the mapped ion pathway through the Na,K-ATPase pump

Science.gov (United States)

Takeuchi, Ayako; Reyes, Nicolás; Artigas, Pablo; Gadsby, David C.

2009-01-01

the Na+,K+-ATPase and by other P-type pumps like the Ca2+- and H+,K+-ATPases. PMID:19806033

Na(+),K (+)-ATPase as a docking station: protein-protein complexes of the Na(+),K (+)-ATPase.

Science.gov (United States)

Reinhard, Linda; Tidow, Henning; Clausen, Michael J; Nissen, Poul

2013-01-01

The Na(+),K(+)-ATPase, or sodium pump, is well known for its role in ion transport across the plasma membrane of animal cells. It carries out the transport of Na(+) ions out of the cell and of K(+) ions into the cell and thus maintains electrolyte and fluid balance. In addition to the fundamental ion-pumping function of the Na(+),K(+)-ATPase, recent work has suggested additional roles for Na(+),K(+)-ATPase in signal transduction and biomembrane structure. Several signaling pathways have been found to involve Na(+),K(+)-ATPase, which serves as a docking station for a fast-growing number of protein interaction partners. In this review, we focus on Na(+),K(+)-ATPase as a signal transducer, but also briefly discuss other Na(+),K(+)-ATPase protein-protein interactions, providing a comprehensive overview of the diverse signaling functions ascribed to this well-known enzyme.

Single-molecule analysis of inhibitory pausing states of V1-ATPase.

Science.gov (United States)

Uner, Naciye Esma; Nishikawa, Yoshihiro; Okuno, Daichi; Nakano, Masahiro; Yokoyama, Ken; Noji, Hiroyuki

2012-08-17

V(1)-ATPase, the hydrophilic V-ATPase domain, is a rotary motor fueled by ATP hydrolysis. Here, we found that Thermus thermophilus V(1)-ATPase shows two types of inhibitory pauses interrupting continuous rotation: a short pause (SP, 4.2 s) that occurred frequently during rotation, and a long inhibitory pause (LP, >30 min) that terminated all active rotations. Both pauses occurred at the same angle for ATP binding and hydrolysis. Kinetic analysis revealed that the time constants of inactivation into and activation from the SP were too short to represent biochemically predicted ADP inhibition, suggesting that SP is a newly identified inhibitory state of V(1)-ATPase. The time constant of inactivation into LP was 17 min, consistent with one of the two time constants governing the inactivation process observed in bulk ATPase assay. When forcibly rotated in the forward direction, V(1) in LP resumed active rotation. Solution ADP suppressed the probability of mechanical activation, suggesting that mechanical rotation enhanced inhibitory ADP release. These features were highly consistent with mechanical activation of ADP-inhibited F(1), suggesting that LP represents the ADP-inhibited state of V(1)-ATPase. Mechanical activation largely depended on the direction and angular displacement of forced rotation, implying that V(1)-ATPase rotation modulates the off rate of ADP.

Expression of FlHMA3, a P1B2-ATPase from Festulolium loliaceum, correlates with response to cadmium stress.

Science.gov (United States)

Guo, Qiang; Meng, Lin; Humphreys, Mike W; Scullion, John; Mur, Luis A J

2017-03-01

Heavy metal ATPase 3 (HMA3), a P 1B2 -ATPase, is a key tonoplast transporter involved in mediating the vacuolar sequestration of cadmium (Cd) to detoxify the intake of this element by plants. HMA3 expression in response to Cd stress has not been previously examined in the grass hybrid species Festulolium loliaceum (Huds.) P. Fourn. In this study, FlHMA3 isolated from F. loliaceum was found to comprise 833 amino acid residues with 77% homology to the rice OsHMA3. Transient expression of FlHMA3 fused to enhanced green fluorescent protein in Arabidopsis protoplasts suggested its localization to vacuolar membranes. Quantitative real-time RT-PCR analysis of F. loliaceum revealed that FlHMA3 is expressed predominantly within roots and up-regulated by excess Cd. Over the 168 h treatment, Cd content of F. loliaceum roots was significantly higher than that of shoots, regardless of external CdCl 2 concentrations. A significant positive correlation was found between FlHMA3 expression and Cd accumulation in roots of F. loliaceum seedlings subjected to 10-100 mg L -1 CdCl 2 for 168 h or, in a separate experiment, to 25 or 100 mg L -1 CdCl 2 for the same duration. These findings provide evidence that FlHMA3 encodes a vacuolar P 1B2 -ATPase that may play an important role in Cd 2+ sequestration into root cell vacuoles, thereby limiting the entry of Cd 2+ into the cytoplasm and reducing Cd 2+ toxicity. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Hibiscus sabdariffa (Roselle) Polyphenol-rich Extract Averts Cardiac Functional and Structural Abnormalities in Type 1 Diabetic Rats.

Science.gov (United States)

Mohammed Yusof, Nur Liyana; Zainalabidin, Satirah; Mohd Fauzi, Norsyahida; Budin, Siti Balkis

2018-05-04

Diabetes mellitus is often associated with cardiac functional and structural alteration, an initial event leading to cardiovascular complications. Hibiscus sabdariffa or roselle has been widely proven as an antioxidant and recently has incited research interest for its potential in treating cardiovascular disease. Therefore, this study aimed to determine the cardioprotective effects of H. sabdariffa (roselle) polyphenol-rich extract (HPE) in type-1 induced diabetic rats. Twenty-four male Sprague-Dawley rats were randomized into four groups (n=6/group): non-diabetic (NDM), diabetic alone (DM), diabetic supplemented with HPE (DM+HPE) and metformin (DM+MET). Type-1 diabetes was induced with streptozotocin (55 mg/kg/i.p). Rats were forced-fed HPE (100 mg/kg) and metformin (150 mg/kg) daily for eight weeks. Results showed that HPE supplementation improved hyperglycemia and dyslipidemia significantly (p<0.05) in DM+HPE compared to DM group. HPE supplementation attenuated cardiac oxidative damage in DM group, indicated by low malondialdehyde and advanced oxidation protein product. As for the antioxidant status, HPE significantly (p<0.05) increased glutathione level, as well as catalase and superoxide dismutase 1 and 2 activities. These findings correlate with cardiac function, whereby HPE supplementation improved left ventricle developed pressure, coronary flow, cardiac contractility and relaxation rate significantly (p<0.05). Histological analysis showed a marked decrease in cardiomyocyte hypertrophy and fibrosis in DM+HPE compared to DM group. Ultrastructural changes and impairment of mitochondria induced by diabetes were minimized by HPE supplementation. Collectively, these findings suggest that HPE is a potential cardioprotective agent in a diabetic setting through its hypoglycemic, anti-hyperlipidemia and antioxidant properties.

Production and characterization of a monoclonal antibody to H+ATPase

International Nuclear Information System (INIS)

Yurko, M.; Fitch, F; Gluck, S.

1986-01-01

Acidification of endocytic vesicles is carried out by an ATP-dependent proton pump, H+ATPase, an FOF1 type enzyme comprised of at least 5 major subunits of 70, 56, 45, 35, and 17 kDa. A monoclonal antibody, H6.1, to H+ATPase from bovine kidney medulla, was raised to enable the structural characterization and localization of the pump. Several criteria were used to show that H6.1 recognized H+ATPase. 1.) H6.1 immunoprecipitated N-ethylmaleimide-sensitive and vanadate- and azide-insensitive solubilized ATPase activity (and GTPase activity) from both crude and purified enzyme preparations. 2.) H6.1 immunoprecipitated oligomycin-insensitive ATP-dependent proton transporting vesicles made from bovine kidney medulla, rat kidney, and CHO cells. 3.) H6.1 specifically immuno-precipitated the 5 subunits of H+ATPase from a partially purified preparation of the enzyme that had been labelled with I-125. H6.1 was then used as an immunocytochemical probe for the localization of H+ATPase. In bovine kidney medullary collecting duct, there was an intense apical staining of selected cells. In proximal tubule and in cultured CHO cells there was a granular pattern of staining characteristic of endocytic vesicles and lysosomes, suggesting that the kidney and CHO cell proton pumps are structurally related

Erythrocyte membrane ATPase and calcium pumping activities in porcine malignant hyperthermia

International Nuclear Information System (INIS)

Thatte, H.S.; Mickelson, J.R.; Addis, P.B.; Louis, C.F.

1987-01-01

To investigate possible abnormalities in erythrocyte membrane enzyme activities in the pharmacogenetic disorder MH, membrane ATPase activities have been examined in erythrocyte ghosts prepared from red blood cells of MHS and normal swine. While no differences were noted in Mg2+-ATPase activities, the (Na+, K+)-ATPase activity of MHS erythrocyte ghosts was less than that of normal ghosts. Ca2+-ATPase activity exhibited low- and high-affinity Ca2+-binding sites in both types of erythrocyte ghost. While the Km for Ca2+ was greater for normal than for MHS erythrocyte ghosts at the high-affinity Ca2+-binding site, the reverse was true at the low-affinity Ca2+-binding site. Irrespective of the type of calcium binding site occupied, the Vmax for normal erythrocyte ghost Ca2+-ATPase activity was greater than that for MHS ghosts. In the presence of calmodulin, there was now no difference between MHS and normal erythrocyte ghosts in either the Km for Ca2+ or the Vmax of the Ca2+-ATPase activity. To determine if the calcium pumping activity of intact MHS and normal pig erythrocytes differed, calcium efflux from the 45 Ca-loaded erythrocytes was determined; this activity was significantly greater for MHS than for normal erythrocytes. Thus, the present study confirms that there are abnormalities in the membranes of MHS pig red blood cells. However, we conclude that these abnormalities are unlikely to result in an impaired ability of MHS erythrocytes to regulate their cytosolic Ca2+ concentration

Effect of ionizing radiation on Ca2+-ATPase and Mg2+-ATPase: the role of ligands

International Nuclear Information System (INIS)

Dreval', V.I.

1994-01-01

The change of Ca 2+ -ATPase and Mg 2+ -ATPase activity in plasma membranes of thymocytes irradiated with doses of 10 2 , 10 3 and 10 4 Gy in the presence of Ca 2+ , Mg 2+ and ATP was studied. Stabilizing effect of Ca 2+ and Mg 2+ on Ca 2+ -ATPase and ATP on Mg 2+ -ATPase under irradiation was established

Effect of TGFβ on Na{sup +}/K{sup +} ATPase activity in megakaryocytes

Energy Technology Data Exchange (ETDEWEB)

Hosseinzadeh, Zohreh; Schmid, Evi; Shumilina, Ekaterina [Department of Physiology, University of Tübingen (Germany); Laufer, Stefan [Pharmaceutical Chemistry, University of Tübingen (Germany); Borst, Oliver; Gawaz, Meinrad [Cardiology and Cardiovascular Medicine, University of Tübingen (Germany); Lang, Florian, E-mail: florian.lang@uni-tuebingen.de [Department of Physiology, University of Tübingen (Germany)

2014-09-26

Highlights: • TGFß1 markedly up-regulates Na{sup +}/K{sup +} ATPase in megakaryocytes. • The effect is abrogated by p38-MAP kinase inhibitor skepinone. • The effect is abrogated by SGK inhibitor EMD638683. • The effect is abrogated by NF-κB inhibitor wogonin. - Abstract: The Na{sup +}/K{sup +} ATPase generates the Na{sup +} and K{sup +} concentration gradients across the plasma membrane and is thus essential for cellular electrolyte homeostasis, cell membrane potential and cell volume maintenance. A powerful regulator of Na{sup +}/K{sup +} ATPase is the serum- and glucocorticoid-inducible kinase 1 (SGK1). The most powerful known regulator of SGK1 expression is TGFß1, which is pivotal in the regulation of megakaryocyte maturation and platelet formation. Signaling involved in the upregulation of SGK1 by TGFß1 includes p38 mitogen activated protein (MAP) kinase. SGK1 in turn phosphorylates the IκB kinase (IKKα/β), which phosphorylates the inhibitor protein IκBα thus triggering nuclear translocation of nuclear factor kappa B (NF-κB). The present study explored whether TGFβ influences Na{sup +}/K{sup +} ATPase activity in megakaryocytes, and if so, whether the effect of TGß1 requires p38 MAP kinase, SGK1 and/or NF-κB. To this end, murine megakaryocytes were treated with TGFß1 and Na{sup +}/K{sup +} ATPase activity determined from K{sup +} induced current utilizing whole cell patch clamp. The pump current (I{sub pump}) was determined in the absence and presence of Na{sup +}/K{sup +} ATPase inhibitor ouabain (100 μM). TGFß1 (60 ng/ml) was added in the absence or presence of p38 MAP kinase inhibitor skepinone-L (1 μM), SGK1 inhibitor EMD638683 (50 μM) or NF-κB inhibitor wogonin (50 nM). As a result, the I{sub pump} was significantly increased by pretreatment of the megakaryocytes with TGFß1, an effect reaching statistical significance within 16 and 24 h and virtually abrogated in the presence of skepinone-L, EMD638683 or wogonin. In conclusion

Loss of the Arabidopsis thaliana P4-ATPases ALA6 and ALA7 Impairs Pollen Fitness and Alters the Pollen Tube Plasma Membrane

Directory of Open Access Journals (Sweden)

Stephen C McDowell

2015-04-01

Full Text Available Members of the P4 subfamily of P-type ATPases are thought to create and maintain lipid asymmetry in biological membranes by flipping specific lipids between membrane leaflets. In Arabidopsis, 7 of the 12 Aminophospholipid ATPase (ALA family members are expressed in pollen. Here we show that double knockout of ALA6 and ALA7 (ala6/7 results in siliques with a ~2-fold reduction in seed set with a high frequency of empty seed positions near the bottom. Seed set was reduced to near zero when plants were grown under a hot/cold temperature stress. Reciprocal crosses indicate that the ala6/7 reproductive deficiencies are due to a defect related to pollen transmission. In-vitro growth assays provide evidence that that ala6/7 pollen tubes are short and slow, with ~2-fold reductions in both maximal growth rate and overall length relative to wild-type. Outcrosses show that when ala6/7 pollen are in competition with wild-type pollen, they have a near 0% success rate in fertilizing ovules near the bottom of the pistil, consistent with ala6/7 pollen having short and slow growth defects. The ala6/7 phenotypes were rescued by the expression of either an ALA6-YFP or GFP-ALA6 fusion protein, which showed localization to both the plasma membrane and highly-mobile endomembrane structures. A mass spectrometry analysis of mature pollen grains revealed significant differences between ala6/7 and wild-type, both in the relative abundance of lipid classes and in the average number of double bonds present in acyl side chains. A change in the properties of the ala6/7 plasma membrane was also indicated by a ~10-fold reduction of labeling by lipophilic FM-dyes relative to wild-type. Together, these results indicate that ALA6 and ALA7 provide redundant activities that function to directly or indirectly change the distribution and abundance lipids in pollen, and support a model in which ALA6 and ALA7 are critical for pollen fitness under normal and temperature-stress conditions.

Myocardial Na,K-ATPase: Clinical aspects

OpenAIRE

Kjeldsen, Keld

2003-01-01

The specific binding of digitalis glycosides to Na,K-ATPase is used as a tool for Na,K-ATPase quantification with high accuracy and precision. In myocardial biopsies from patients with heart failure, total Na,K-ATPase concentration is decreased by around 40%; a correlation exists between a decrease in heart function and a decrease in Na,K-ATPase concentration. During digitalization, around 30% of remaining pumps are occupied by digoxin. Myocardial Na,K-ATPase is also influenced by other drugs...

Left atrial dysfunction in type 2 diabetes mellitus: insights from cardiac MRI

Energy Technology Data Exchange (ETDEWEB)

Graca, Bruno; Donato, Paulo; Caseiro-Alves, Filipe [University of Coimbra, Faculty of Medicine, Coimbra (Portugal); Coimbra' s Hospital Centre and University, Medical Imaging Department, Coimbra (Portugal); Joao Ferreira, Maria [University of Coimbra, Faculty of Medicine, Coimbra (Portugal); Coimbra' s Hospital Centre and University, Cardiology Department, Coimbra (Portugal); Gomes, Leonor [University of Coimbra, Faculty of Medicine, Coimbra (Portugal); Coimbra' s Hospital Centre and University, Endocrinology Department, Coimbra (Portugal); Castelo-Branco, Miguel [University of Coimbra, Faculty of Medicine, Coimbra (Portugal)

2014-11-15

The left atrium (LA) modulates left ventricular filling through reservoir, conduit and booster pump functions. Only limited data exist on LA involvement in type 2 diabetes mellitus (DM2). This study sought to assess LA function in asymptomatic DM2 with cardiac MRI. We hypothesized that cardiac MRI can detect LA dysfunction in asymptomatic DM2. Forty-five patients with asymptomatic DM2 and 24 normoglycaemic controls were studied. MRI cine imaging was performed to measure LA maximal and minimal volumes. A flow-sensitive phase-contrast gradient-echo sequence was used for flow measurements perpendicular to the orifice of the mitral valve, to quantify active LA stroke volume. LA total, passive and active emptying volumes and fractions were calculated. LA reservoir function, namely LA total ejection fraction, was significantly greater in controls compared to patients with DM2 (62.2 ± 5.2 vs 57.0 ± 7.6 %, P = 0.004). LA passive ejection fraction was also greater in the controls (26.2 ± 9.5 vs 16.1 ± 11.0 %, P < 0.001). Regarding parameters of LA booster pump function, LA active ejection fraction was not significantly different between groups. DM2 was demonstrated to be an independent determinant of LA function. Cardiac MRI enables the detection of LA dysfunction in asymptomatic DM2, characterized by a reduction in LA reservoir and conduit functions. (orig.)

Aspirin suppresses cardiac fibroblast proliferation and collagen formation through downregulation of angiotensin type 1 receptor transcription

International Nuclear Information System (INIS)

Wang, Xianwei; Lu, Jingjun; Khaidakov, Magomed; Mitra, Sona; Ding, Zufeng; Raina, Sameer; Goyal, Tanu; Mehta, Jawahar L.

2012-01-01

Aspirin (acetyl salicylic acid, ASA) is a common drug used for its analgesic and antipyretic effects. Recent studies show that ASA not only blocks cyclooxygenase, but also inhibits NADPH oxidase and resultant reactive oxygen species (ROS) generation, a pathway that underlies pathogenesis of several ailments, including hypertension and tissue remodeling after injury. In these disease states, angiotensin II (Ang II) activates NADPH oxidase via its type 1 receptor (AT1R) and leads to fibroblast growth and collagen synthesis. In this study, we examined if ASA would inhibit NADPH oxidase activation, upregulation of AT1R transcription, and subsequent collagen generation in mouse cardiac fibroblasts challenged with Ang II. Mouse heart fibroblasts were isolated and treated with Ang II with or without ASA. As expected, Ang II induced AT1R expression, and stimulated cardiac fibroblast growth and collagen synthesis. The AT1R blocker losartan attenuated these effects of Ang II. Similarly to losartan, ASA, and its SA moiety suppressed Ang II-mediated AT1R transcription and fibroblast proliferation as well as expression of collagens and MMPs. ASA also suppressed the expression of NADPH oxidase subunits (p22 phox , p47 phox , p67 phox , NOX2 and NOX4) and ROS generation. ASA did not affect total NF-κB p65, but inhibited its phosphorylation and activation. These observations suggest that ASA inhibits Ang II-induced NADPH oxidase expression, NF-κB activation and AT1R transcription in cardiac fibroblasts, and fibroblast proliferation and collagen expression. The critical role of NADPH oxidase activity in stimulation of AT1R transcription became apparent in experiments where ASA also inhibited AT1R transcription in cardiac fibroblasts challenged with H 2 O 2 . Since SA had similar effect as ASA on AT1R expression, we suggest that ASA's effect is mediated by its SA moiety. -- Highlights: ► Aspirin in therapeutic concentrations decreases mouse cardiac fibroblast growth and collagen

Aspirin suppresses cardiac fibroblast proliferation and collagen formation through downregulation of angiotensin type 1 receptor transcription

Energy Technology Data Exchange (ETDEWEB)

Wang, Xianwei, E-mail: XWang2@UAMS.edu; Lu, Jingjun; Khaidakov, Magomed; Mitra, Sona; Ding, Zufeng; Raina, Sameer; Goyal, Tanu; Mehta, Jawahar L., E-mail: MehtaJL@UAMS.edu

2012-03-15

Aspirin (acetyl salicylic acid, ASA) is a common drug used for its analgesic and antipyretic effects. Recent studies show that ASA not only blocks cyclooxygenase, but also inhibits NADPH oxidase and resultant reactive oxygen species (ROS) generation, a pathway that underlies pathogenesis of several ailments, including hypertension and tissue remodeling after injury. In these disease states, angiotensin II (Ang II) activates NADPH oxidase via its type 1 receptor (AT1R) and leads to fibroblast growth and collagen synthesis. In this study, we examined if ASA would inhibit NADPH oxidase activation, upregulation of AT1R transcription, and subsequent collagen generation in mouse cardiac fibroblasts challenged with Ang II. Mouse heart fibroblasts were isolated and treated with Ang II with or without ASA. As expected, Ang II induced AT1R expression, and stimulated cardiac fibroblast growth and collagen synthesis. The AT1R blocker losartan attenuated these effects of Ang II. Similarly to losartan, ASA, and its SA moiety suppressed Ang II-mediated AT1R transcription and fibroblast proliferation as well as expression of collagens and MMPs. ASA also suppressed the expression of NADPH oxidase subunits (p22{sup phox}, p47{sup phox}, p67{sup phox}, NOX2 and NOX4) and ROS generation. ASA did not affect total NF-κB p65, but inhibited its phosphorylation and activation. These observations suggest that ASA inhibits Ang II-induced NADPH oxidase expression, NF-κB activation and AT1R transcription in cardiac fibroblasts, and fibroblast proliferation and collagen expression. The critical role of NADPH oxidase activity in stimulation of AT1R transcription became apparent in experiments where ASA also inhibited AT1R transcription in cardiac fibroblasts challenged with H{sub 2}O{sub 2}. Since SA had similar effect as ASA on AT1R expression, we suggest that ASA's effect is mediated by its SA moiety. -- Highlights: ► Aspirin in therapeutic concentrations decreases mouse cardiac

Meiotic Clade AAA ATPases: Protein Polymer Disassembly Machines.

Science.gov (United States)

Monroe, Nicole; Hill, Christopher P

2016-05-08

Meiotic clade AAA ATPases (ATPases associated with diverse cellular activities), which were initially grouped on the basis of phylogenetic classification of their AAA ATPase cassette, include four relatively well characterized family members, Vps4, spastin, katanin and fidgetin. These enzymes all function to disassemble specific polymeric protein structures, with Vps4 disassembling the ESCRT-III polymers that are central to the many membrane-remodeling activities of the ESCRT (endosomal sorting complexes required for transport) pathway and spastin, katanin p60 and fidgetin affecting multiple aspects of cellular dynamics by severing microtubules. They share a common domain architecture that features an N-terminal MIT (microtubule interacting and trafficking) domain followed by a single AAA ATPase cassette. Meiotic clade AAA ATPases function as hexamers that can cycle between the active assembly and inactive monomers/dimers in a regulated process, and they appear to disassemble their polymeric substrates by translocating subunits through the central pore of their hexameric ring. Recent studies with Vps4 have shown that nucleotide-induced asymmetry is a requirement for substrate binding to the pore loops and that recruitment to the protein lattice via MIT domains also relieves autoinhibition and primes the AAA ATPase cassettes for substrate binding. The most striking, unifying feature of meiotic clade AAA ATPases may be their MIT domain, which is a module that is found in a wide variety of proteins that localize to ESCRT-III polymers. Spastin also displays an adjacent microtubule binding sequence, and the presence of both ESCRT-III and microtubule binding elements may underlie the recent findings that the ESCRT-III disassembly function of Vps4 and the microtubule-severing function of spastin, as well as potentially katanin and fidgetin, are highly coordinated. Copyright © 2015 Elsevier Ltd. All rights reserved.

Targeting vacuolar H+-ATPases as a new strategy against cancer.

Science.gov (United States)

Fais, Stefano; De Milito, Angelo; You, Haiyan; Qin, Wenxin

2007-11-15

Growing evidence suggests a key role of tumor acidic microenvironment in cancer development, progression, and metastasis. As a consequence, the need for compounds that specifically target the mechanism(s) responsible for the low pH of tumors is increasing. Among the key regulators of the tumor acidic microenvironment, vacuolar H(+)-ATPases (V-ATPases) play an important role. These proteins cover a number of functions in a variety of normal as well as tumor cells, in which they pump ions across the membranes. We discuss here some recent results showing that a molecular inhibition of V-ATPases by small interfering RNA in vivo as well as a pharmacologic inhibition through proton pump inhibitors led to tumor cytotoxicity and marked inhibition of human tumor growth in xenograft models. These results propose V-ATPases as a key target for new strategies in cancer treatment.

Characterization of the vacuolar H sup + -ATPase of higher plants

Energy Technology Data Exchange (ETDEWEB)

Manolson, M F

1988-01-01

The tonoplast H{sup +}-ATPase of Beta vulgaris L. was partially purified by Triton X-100 solubilization and Sepharose 4B chromatography resulting in the enrichment of two polypeptides. Kinetic analysis of ({alpha}-{sup 32}P) BzATP labeling identified the 57 kDa polypeptide as a nucleotide-binding subunit with a possible regulatory function. In addition, ({sup 14}C) DCCD-labeling identified a 16 kDa polypeptide as a putative transmembrane proton channel. It is concluded that the tonoplast H{sup +}-ATPase is a multimer composed of at least three polypeptides. Anti-57 and anti-67 kDa sera reacted with polypeptides of the corresponding size in bovine chromaffin granules, bovine clathrin-coated vesicles, and yeast vacuolar membranes, suggesting common structural features and common ancestry for endomembrane H{sup +}-ATPase of different organelles and different phyla. Anti-57 serum was used to isolate a cDNA encoding the corresponding subunit from Arabidopsis. Protein sequence analysis revealed homologies between endomembrane, F{sub 0}F{sub 1} and archaebacterial ATPases, suggesting that these different classes of ATPases have evolved from a common ancestor.

Environmental and Genetic Factors Regulating Localization of the Plant Plasma Membrane H+-ATPase.

Science.gov (United States)

Haruta, Miyoshi; Tan, Li Xuan; Bushey, Daniel B; Swanson, Sarah J; Sussman, Michael R

2018-01-01

A P-type H + -ATPase is the primary transporter that converts ATP to electrochemical energy at the plasma membrane of higher plants. Its product, the proton-motive force, is composed of an electrical potential and a pH gradient. Many studies have demonstrated that this proton-motive force not only drives the secondary transporters required for nutrient uptake, but also plays a direct role in regulating cell expansion. Here, we have generated a transgenic Arabidopsis ( Arabidopsis thaliana ) plant expressing H + -ATPase isoform 2 (AHA2) that is translationally fused with a fluorescent protein and examined its cellular localization by live-cell microscopy. Using a 3D imaging approach with seedlings grown for various times under a variety of light intensities, we demonstrate that AHA2 localization at the plasma membrane of root cells requires light. In dim light conditions, AHA2 is found in intracellular compartments, in addition to the plasma membrane. This localization profile was age-dependent and specific to cell types found in the transition zone located between the meristem and elongation zones. The accumulation of AHA2 in intracellular compartments is consistent with reduced H + secretion near the transition zone and the suppression of root growth. By examining AHA2 localization in a knockout mutant of a receptor protein kinase, FERONIA, we found that the intracellular accumulation of AHA2 in the transition zone is dependent on a functional FERONIA-dependent inhibitory response in root elongation. Overall, this study provides a molecular underpinning for understanding the genetic, environmental, and developmental factors influencing root growth via localization of the plasma membrane H + -ATPase. © 2018 American Society of Plant Biologists. All Rights Reserved.

Roles of transmembrane segment M1 of Na(+),K (+)-ATPase and Ca (2+)-ATPase, the gatekeeper and the pivot

DEFF Research Database (Denmark)

Einholm, Anja P.; Andersen, Jens Peter; Vilsen, Bente

2007-01-01

In this review we summarize mutagenesis work on the structure-function relationship of transmembrane segment M1 in the Na(+),K(+)-ATPase and the sarco(endo)plasmic reticulum Ca(2+)-ATPase. The original hypothesis that charged residues in the N-terminal part of M1 interact with the transported...... cations can be rejected. On the other hand hydrophobic residues in the middle part of M1 turned out to play crucial roles in Ca(2+) interaction/occlusion in Ca(2+)-ATPase and K(+) interaction/occlusion in Na(+),K(+)-ATPase. Leu(65) of the Ca(2+)-ATPase and Leu(99) of the Na(+),K(+)-ATPase, located...... of the extracytoplasmic gate in both the Ca(2+)-ATPase and the Na(+),K(+)-ATPase. Udgivelsesdato: 2007-Dec...

Type D patients report poorer health status prior to and after cardiac rehabilitation compared to non-type D patients

OpenAIRE

Pelle, Aline; Erdman, Ruud; Domburg, Ron; Spiering, Marquita; Kazemier, Marten; Pedersen, Susanne

2008-01-01

textabstractBackground: Type D personality is an emerging risk factor in coronary artery disease (CAD). Cardiac rehabilitation (CR) improves outcomes, but little is known about the effects of CR on Type D patients. Purpose: We examined (1) variability in Type D caseness following CR, (2) Type D as a determinant of health status, and (3) the clinical relevance of Type D as a determinant of health status compared to cardiac history. Methods: CAD patients (n = 368) participating in CR completed ...

Na,K-ATPase structure/function relationships probed by the denaturant urea.

Science.gov (United States)

Esmann, Mikael; Fedosova, Natalya U; Olesen, Claus

2015-05-01

Urea interacts with the Na,K-ATPase, leading to reversible as well as irreversible inhibition of the hydrolytic activity. The enzyme purified from shark rectal glands is more sensitive to urea than Na,K-ATPase purified from pig kidney. An immediate and reversible inhibition under steady-state conditions of hydrolytic activity at 37°C is demonstrated for the three reactions studied: the overall Na,K-ATPase activity, the Na-ATPase activity observed in the absence of K+ as well as the K+-dependent phosphatase reaction (K-pNPPase) seen in the absence of Na+. Half-maximal inhibition is seen with about 1M urea for shark enzyme and about 2M urea for pig enzyme. In the presence of substrates there is also an irreversible inhibition in addition to the reversible process, and we show that ATP protects against the irreversible inhibition for both the Na,K-ATPase and Na-ATPase reaction, whereas the substrate paranitrophenylphosphate leads to a slight increase in the rate of irreversible inhibition of the K-pNPPase. The rate of the irreversible inactivation in the absence of substrates is much more rapid for shark enzyme than for pig enzyme. The larger number of potentially urea-sensitive hydrogen bonds in shark enzyme compared to pig enzyme suggests that interference with the extensive hydrogen bonding network might account for the higher urea sensitivity of shark enzyme. The reversible inactivation is interpreted in terms of domain interactions and domain accessibilities using as templates the available crystal structures of Na,K-ATPase. It is suggested that a few interdomain hydrogen bonds are those mainly affected by urea during reversible inactivation. Copyright © 2014 Elsevier B.V. All rights reserved.

{sup 123}I-MIBG imaging detects cardiac involvement and predicts cardiac events in Churg-Strauss syndrome

Energy Technology Data Exchange (ETDEWEB)

Horiguchi, Yoriko; Morita, Yukiko [National Hospital Organization Sagamihara National Hospital, Department of Cardiology, Sagamihara City, Kanagawa (Japan); Tsurikisawa, Naomi; Akiyama, Kazuo [National Hospital Organization Sagamihara National Hospital, Clinical Research Centre for Allergy and Rheumatology, Sagamihara City, Kanagawa (Japan)

2011-02-15

In Churg-Strauss syndrome (CSS) it is important to detect cardiac involvement, which predicts poor prognosis. This study evaluated whether {sup 123}I-metaiodobenzylguanidine (MIBG) scintigraphy could detect cardiac damage and predict cardiac events in CSS. {sup 123}I-MIBG scintigraphy was performed in 28 patients with CSS, 12 of whom had cardiac involvement. The early and delayed heart to mediastinum ratio (early H/M and delayed H/M) and washout rate were calculated by using {sup 123}I-MIBG scintigraphy and compared with those in control subjects. Early H/M and delayed H/M were significantly lower and the washout rate was significantly higher in patients with cardiac involvement than in those without and in controls (early H/M, p = 0.0024, p = 0.0001; delayed H/M, p = 0.0002, p = 0.0001; washout rate, p = 0.0012, p = 0.0052 vs those without and vs controls, respectively). Accuracy for detecting cardiac involvement was 86% for delayed H/M and washout rate and 79% for early H/M and B-type natriuretic peptide (BNP). Kaplan-Meier analysis showed significantly lower cardiac event-free rates in patients with early H/M {<=} 2.18 and BNP > 21.8 pg/ml than those with early H/M > 2.18 and BNP {<=} 21.8 pg/ml (log-rank test p = 0.006). Cardiac sympathetic nerve function was damaged in CSS patients with cardiac involvement. {sup 123}I-MIBG scintigraphy was useful in detecting cardiac involvement and in predicting cardiac events. (orig.)

[Properties and localization of Mg- and Ca-ATpase activities in wheat embryo cell nuclei].

Science.gov (United States)

Vasil'eva, N A; Belkina, G G; Stepanenko, S Y; Atalykova, F I; Oparin, A I

1978-05-01

The isolated nuclei of wheat embryo possess the ATPase activity. The addition of Mg2+ and Ca2+ significantly increases the activities of nuclear ATPases, whereas Hg2+, Cu2+ and Mn2+ inhibit the activity. The activating effect of Mg2+ is enhanced by an addition of Na and K ions. The activity of wheat embryo nuclear Mg-ATPase is higher than its Ca-ATPase activity; both ATPases also differ in their pH optima. Separation of total nuclear protein according to the solubility of its individual protein components in wheat and strong salt solutions, using the detergents, as well as ammonium sulfate precipitation and dialysis do not result in separation of Mg-activated and Ca-activated ATPases, although their levels of activities and ratios change in the course of fractionation. The Mg- and Ca-ATPase activities of the wheat embryo nuclei were found in the nuclear fraction of albumin, in nonhistone proteins and nuclear membranes. In the albumin nuclear fraction and subfractions of non-histone proteins the higher level of activity is observed in Ca-ATPase, whereas in the nuclei and soluble fractions of residual proteins in Mg-ATPase.

Gender Differences and Demographics and Type of Cardiac Device ...

African Journals Online (AJOL)

2018-02-07

Feb 7, 2018 ... relation between gender and cardiac device implantation indications and ... cerebrovascular disease, DM = diabetes mellitus, HT = hypertension,. PAD = peripheral arterial ... Figure 2: Trend of device types according to years.

Na+-stimulated ATPase of alkaliphilic halotolerant cyanobacterium Aphanothece halophytica translocates Na+ into proteoliposomes via Na+ uniport mechanism

Directory of Open Access Journals (Sweden)

Soontharapirakkul Kanteera

2010-08-01

Full Text Available Abstract Background When cells are exposed to high salinity conditions, they develop a mechanism to extrude excess Na+ from cells to maintain the cytoplasmic Na+ concentration. Until now, the ATPase involved in Na+ transport in cyanobacteria has not been characterized. Here, the characterization of ATPase and its role in Na+ transport of alkaliphilic halotolerant Aphanothece halophytica were investigated to understand the survival mechanism of A. halophytica under high salinity conditions. Results The purified enzyme catalyzed the hydrolysis of ATP in the presence of Na+ but not K+, Li+ and Ca2+. The apparent Km values for Na+ and ATP were 2.0 and 1.2 mM, respectively. The enzyme is likely the F1F0-ATPase based on the usual subunit pattern and the protection against N,N'-dicyclohexylcarbodiimide inhibition of ATPase activity by Na+ in a pH-dependent manner. Proteoliposomes reconstituted with the purified enzyme could take up Na+ upon the addition of ATP. The apparent Km values for this uptake were 3.3 and 0.5 mM for Na+ and ATP, respectively. The mechanism of Na+ transport mediated by Na+-stimulated ATPase in A. halophytica was revealed. Using acridine orange as a probe, alkalization of the lumen of proteoliposomes reconstituted with Na+-stimulated ATPase was observed upon the addition of ATP with Na+ but not with K+, Li+ and Ca2+. The Na+- and ATP-dependent alkalization of the proteoliposome lumen was stimulated by carbonyl cyanide m - chlorophenylhydrazone (CCCP but was inhibited by a permeant anion nitrate. The proteoliposomes showed both ATPase activity and ATP-dependent Na+ uptake activity. The uptake of Na+ was enhanced by CCCP and nitrate. On the other hand, both CCCP and nitrate were shown to dissipate the preformed electric potential generated by Na+-stimulated ATPase of the proteoliposomes. Conclusion The data demonstrate that Na+-stimulated ATPase from A. halophytica, a likely member of F-type ATPase, functions as an electrogenic Na

On archaebacterial ATPase from Halobacterium saccharovorum

Science.gov (United States)

Kristjansson, H.; Ponnamperuma, C.; Hochstein, L.; Altekar, W.

1984-01-01

The energy transducing ATPase from Halobacterium saccharovorum was studied in order to define the origin of energy transducing systems. The ATPase required high salt concentration (4M NaCl) for activity; activity was rapidly lost when NaCl was below 1 Molar. At low salt concentration, the membrane bound ATPase activity could be stabilized in presence of spermine. However, following solubilization spermine was ineffective. Furthermore, F1 ATPase activity was stabilized by ammonium sulfate even when the NaCl concentration was less than 1 Molar. These studies suggest that stabilization by hydrophobic interactions preceded ionic ones in the evolution of the energy transducing ATPases.

Overexpression of Sarcoendoplasmic Reticulum Calcium ATPase 2a Promotes Cardiac Sympathetic Neurotransmission via Abnormal Endoplasmic Reticulum and Mitochondria Ca2+ Regulation

Science.gov (United States)

Shanks, Julia; Herring, Neil; Johnson, Errin; Liu, Kun; Li, Dan

2017-01-01

Reduced cardiomyocyte excitation–contraction coupling and downregulation of the SERCA2a (sarcoendoplasmic reticulum calcium ATPase 2a) is associated with heart failure. This has led to viral transgene upregulation of SERCA2a in cardiomyocytes as a treatment. We hypothesized that SERCA2a gene therapy expressed under a similar promiscuous cytomegalovirus promoter could also affect the cardiac sympathetic neural axis and promote sympathoexcitation. Stellate neurons were isolated from 90 to 120 g male, Sprague–Dawley, Wistar Kyoto, and spontaneously hypertensive rats. Neurons were infected with Ad-mCherry or Ad-mCherry-hATP2Aa (SERCA2a). Intracellular Ca2+ changes were measured using fura-2AM in response to KCl, caffeine, thapsigargin, and carbonylcyanide-p-trifluoromethoxyphenylhydrazine to mobilize intracellular Ca2+ stores. The effect of SERCA2a on neurotransmitter release was measured using [3H]-norepinephrine overflow from 340 to 360 g Sprague–Dawley rat atria in response to right stellate ganglia stimulation. Upregulation of SERCA2a resulted in greater neurotransmitter release in response to stellate stimulation compared with control (empty: 98.7±20.5 cpm, n=7; SERCA: 186.5±28.41 cpm, n=8; Pneurons, SERCA2a overexpression facilitated greater depolarization-induced Ca2+ transients (empty: 0.64±0.03 au, n=57; SERCA: 0.75±0.03 au, n=68; Pneurons resulted in increased neurotransmission and increased Ca2+ loading into intracellular stores. Whether the increased Ca2+ transient and neurotransmission after SERCA2A overexpression contributes to enhanced sympathoexcitation in heart failure patients remains to be determined. PMID:28223472

Active site-directed alkylation of Na+-K+-ATPase by digitalis sulphonate derivatives of different lipophilicity.

Science.gov (United States)

Fricke, U.; Klaus, W.; Rogatti, M.

1981-01-01

1 Sulphonate derivatives of k-strophanthidin and digitoxigenin were tested as active site-directed labels of Na+-K+-adenosine triphosphatase (Na+-ATPase) from guinea-pig heart. 2 Lipophilicity ranged between P = 93 for strophanthidin-3-tosyloxy-acetate (STA) and P = 3028 for digitoxigenin-3-tosyloxy-acetate (DTA). 3 Although the alkylating moiety of STA and DTA was identical, the reversibility of Na+-K+-ATPase inhibition varied appreciably (82% and 35% respectively). 4 It is concluded that lipophilicity contributes considerably to the irreversible binding of alkylating cardiotonic steroids to myocardial Na+-K+-ATPase. PMID:6261865

Binding of ouabain and marinobufagenin leads to different structural changes in Na,K-ATPase and depends on the enzyme conformation.

Science.gov (United States)

Klimanova, Elizaveta A; Petrushanko, Irina Yu; Mitkevich, Vladimir A; Anashkina, Anastasia A; Orlov, Sergey N; Makarov, Alexander A; Lopina, Olga D

2015-09-14

Ion pump, Na,K-ATPase specifically binds cardiotonic steroids (CTS), which leads to inhibition of the enzyme activity and activation of signaling network in the cell. We have studied interaction of Na,K-ATPase with CTS of two different types - marinobufagenin and ouabain. We have shown that both CTS inhibit activity of Na,K-ATPase with the same Ki values, but binding of ouabain is sensitive to the conformation of Na,K-ATPase while binding of marinobufagenin is not. Furthermore, binding of ouabain and marinobufagenin results in different structural changes in Na,K-ATPase. Our data allow to explain the diversity of effects on the receptor function of Na,K-ATPase caused by different types of CTS. Copyright © 2015 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Identification of V-ATPase as a molecular sensor of SOX11-levels and potential therapeutic target for mantle cell lymphoma

International Nuclear Information System (INIS)

Emruli, Venera Kuci; Olsson, Roger; Ek, Fredrik; Ek, Sara

2016-01-01

Mantle cell lymphoma (MCL) is an aggressive disease with short median survival. Molecularly, MCL is defined by the t(11;14) translocation leading to overexpression of the CCND1 gene. However, recent data show that the neural transcription factor SOX11 is a disease defining antigen and several involved signaling pathways have been pin-pointed, among others the Wnt/β-catenin pathway that is of importance for proliferation in MCL. Therefore, we evaluated a compound library focused on the Wnt pathway with the aim of identifying Wnt-related targets that regulate growth and survival in MCL, with particular focus on SOX11-dependent growth regulation. An inducible SOX11 knock-down system was used to functionally screen a library of compounds (n = 75) targeting the Wnt signaling pathway. A functionally interesting target, vacuolar-type H + -ATPase (V-ATPase), was further evaluated by western blot, siRNA-mediated gene silencing, immunofluorescence, and flow cytometry. We show that 15 out of 75 compounds targeting the Wnt pathway reduce proliferation in all three MCL cell lines tested. Furthermore, three substances targeting two different targets (V-ATPase and Dkk1) showed SOX11-dependent activity. Further validation analyses were focused on V-ATPase and showed that two independent V-ATPase inhibitors (bafilomycin A1 and concanamycin A) are sensitive to SOX11 levels, causing reduced anti-proliferative response in SOX11 low cells. We further show, using fluorescence imaging and flow cytometry, that V-ATPase is mainly localized to the plasma membrane in primary and MCL cell lines. We show that SOX11 status affect V-ATPase dependent pathways, and thus may be involved in regulating pH in intracellular and extracellular compartments. The plasma membrane localization of V-ATPase indicates that pH regulation of the immediate extracellular compartment may be of importance for receptor functionality and potentially invasiveness in vivo. The online version of this article (doi:10

Structures and immunolocalization of Na+, K+ -ATPase, Na+ /H+ exchanger 3 and vacuolar-type H+ -ATPase in the gills of blennies (Teleostei: Blenniidae) inhabiting rocky intertidal areas.

Science.gov (United States)

Uchiyama, M; Komiyama, M; Yoshizawa, H; Shimizu, N; Konno, N; Matsuda, K

2012-05-01

The structure and immunolocalization of the ion transporters Na(+) ,K(+) -ATPase (NKA), Na(+) /H(+) exchanger (NHE3) and vacuolar-type H(+) -ATPase (VHA) were examined in the gills of teleosts of the family Blenniidae, which inhabit rocky shores with vertical zonation in subtropical seas. These features were compared among the following species with different ecologies: the amphibious rockskipper blenny Andamia tetradactylus, the intertidal white-finned blenny Praealticus tanegasimae and the purely marine yaeyama blenny Ecsenius yaeyamaensis. Light and electron microscopic observations indicated that thick gill filaments were arranged close to each other and alternately on two hemibranches of a gill arch in the opercular space of A. tetradactylus. Many mucous cells (MC) and mitochondrion-rich cells (MRC) were present in the interlamellar regions of the gill filament. An immunohistochemical study demonstrated that numerous NKA, NHE3 and some VHA were located predominantly on presumed MRCs of gill filaments and at the base of the lamellae. Analyses using serial (mirror image) sections of the gills indicated that only a few NKA immunoreactive cells (IRC) were colocalized with VHA on some MRCs in the filaments. In the gills of P. tanegasimae, NKA- and NHE3-IRCs were observed in the interlamellar region of the filaments and at the base of the lamellae. VHA-IRCs were located sparsely on the lamellae and filaments. In the gills of E. yaeyamaensis, the lamellae and filaments were thin and straight, respectively. MCs were located at the tip as well as found scattered in the interlamellar region of gill filaments. NKA-, NHE3- and VHA-IRCs were moderately frequently observed in the filaments and rarely on the lamellae. This study shows that the structure and distribution of ion transporters in the gills differ among the three blennid species, presumably reflecting their different ecologies. © 2012 The Authors. Journal of Fish Biology © 2012 The Fisheries Society of the

Protein-protein interactions within the ensemble, eukaryotic V-ATPase, and its concerted interactions with cellular machineries.

Science.gov (United States)

Balakrishna, Asha Manikkoth; Manimekalai, Malathy Sony Subramanian; Grüber, Gerhard

2015-10-01

The V1VO-ATPase (V-ATPase) is the important proton-pump in eukaryotic cells, responsible for pH-homeostasis, pH-sensing and amino acid sensing, and therefore essential for cell growths and metabolism. ATP-cleavage in the catalytic A3B3-hexamer of V1 has to be communicated via several so-called central and peripheral stalk units to the proton-pumping VO-part, which is membrane-embedded. A unique feature of V1VO-ATPase regulation is its reversible disassembly of the V1 and VO domain. Actin provides a network to hold the V1 in proximity to the VO, enabling effective V1VO-assembly to occur. Besides binding to actin, the 14-subunit V-ATPase interacts with multi-subunit machineries to form cellular sensors, which regulate the pH in cellular compartments or amino acid signaling in lysosomes. Here we describe a variety of subunit-subunit interactions within the V-ATPase enzyme during catalysis and its protein-protein assembling with key cellular machineries, essential for cellular function. Copyright © 2015 Elsevier Ltd. All rights reserved.

Increasing acidification of nonreplicating Lactococcus lactis Delta thyA mutants by incorporating ATPase activity

DEFF Research Database (Denmark)

Pedersen, Martin Bastian; Købmann, Brian Jensen; Jensen, Peter Ruhdal

2002-01-01

% of that of exponentially growing MBP71. However, when nonspecific ATPase activity was incorporated into MBP71, the lactic acid flux was restored to 100% but not above that point, indicating that control over the flux switched from ATP demand to ATP supply (i.e., to sugar transport and glycolysis). As determined by growing...... nonreplicating cells with high ATPase activity on various sugar sources, it appeared that glycolysis exerted the majority of the control. ATPase activity also stimulated the rate of acidification by noureplicating MBP71 growing in milk, and pH 5.2 was reached 40% faster than it was without ATPase activity. We...... concluded that ATPase activity is a functional means of increasing acidification by nonreplicating L. lactis....

Structures and characterization of digoxin- and bufalin-bound Na+,K+-ATPase compared with the ouabain-bound complex

DEFF Research Database (Denmark)

Laursen, Mette; Gregersen, Jonas Lindholt; Yatime, Laure

2015-01-01

. These functionalities have specific influence on the binding properties. We report crystal structures of the Na+,K+-ATPase in the E2P form in complex with bufalin (a nonglycosylated CTS with a six-membered lactone) and digoxin (a trisaccharide-conjugated CTS with a five-membered lactone) and compare......Cardiotonic steroids (CTSs) are specific and potent inhibitors of the Na+,K+-ATPase, with highest affinity to the phosphoenzyme (E2P) forms. CTSs are comprised of a steroid core, which can be glycosylated, and a varying number of substituents, including a five- or six-membered lactone...... in site II of the digoxin and ouabain complexes, whereas both sites are occupied by K+ in the E2P–bufalin complex. In all complexes, αM4 adopts a wound form, characteristic for the E2P state and favorable for high-affinity CTS binding. We conclude that the occupants of the cation-binding site and the type...

Modelled microgravity alters the Na+, K+-ATPase activity in rat heart homogenates

Science.gov (United States)

Peana, Alessandra T.; Pippia, Proto; Paci, Silvia; Tognacini, Christina; Assaretti, Anna Rita; Meloni, Antonietta M.; Galleri, Grazia; Bernardini, Federico

2005-08-01

This study was aimed at establishing whether modeled microgravity conditions, created in a three-dimensional clinostat (Random Positioning Machine, RPM), influence the membrane-associated Na+, K+- and Mg2+- ATPase activities in heart homogenates from rats (ex- posed to RPM for 48 hours). The experimental data indicate that modeled low g significantly decreased the total ATPase (p<0.01) and Na+, K+ -ATPase activities (p<0.05) with no change of the Mg2+-ATPase activity, compared to the respective rat control groups (ground). This Na+, K+- pump inhibition could cause a digital- like effect in response to several modifications of many physiological processes even if this inhibition might also be causally related to the physiological environment induced by RPM. The exact mechanism by which total A TPase and Na+, K+ -A TPase activities decrease in response to RPM conditions remains to be established. We cannot rule out that a reduced intracellular ATP production, previously demonstrated in other cellular systems submitted to modeled microgravity conditions, could be responsible for the effects reported here.

Radionuclide assay of membrane Na+, K+-ATPase activity of peserved red blood cells

International Nuclear Information System (INIS)

Trusov, V.V.; Zelenin, A.A.; Marizin, S.A.

1986-01-01

The radionuclide tests were used to investigate the influence of varying blood preservatives on erythrocylic membrane Na + , K + -ATPase activity in samples of whole blood and packed red blood cells from normal donors prepared by standard methods. The tests were performed before and after seven days of preservation under standard conditions. It was found that blood preservations lowered membrane Na + , K + -ATPase activity: its minimum reduction was recorded with citroglucopnosphate, while glugicir induced a significant drop in Na + , K + -ATPase activity of preserved red blood cells regardless of the type of the blood transfusion solution. The assay of membrane Na + , K + -ATPase activity of preserved red blood cells with the use of 86 Rb could be recommended as an evaluation test for preserved blood and its components

Purification, characterization and crystallization of the F-ATPase from Paracoccus denitrificans.

Science.gov (United States)

Morales-Rios, Edgar; Watt, Ian N; Zhang, Qifeng; Ding, Shujing; Fearnley, Ian M; Montgomery, Martin G; Wakelam, Michael J O; Walker, John E

2015-09-01

The structures of F-ATPases have been determined predominantly with mitochondrial enzymes, but hitherto no F-ATPase has been crystallized intact. A high-resolution model of the bovine enzyme built up from separate sub-structures determined by X-ray crystallography contains about 85% of the entire complex, but it lacks a crucial region that provides a transmembrane proton pathway involved in the generation of the rotary mechanism that drives the synthesis of ATP. Here the isolation, characterization and crystallization of an integral F-ATPase complex from the α-proteobacterium Paracoccus denitrificans are described. Unlike many eubacterial F-ATPases, which can both synthesize and hydrolyse ATP, the P. denitrificans enzyme can only carry out the synthetic reaction. The mechanism of inhibition of its ATP hydrolytic activity involves a ζ inhibitor protein, which binds to the catalytic F₁-domain of the enzyme. The complex that has been crystallized, and the crystals themselves, contain the nine core proteins of the complete F-ATPase complex plus the ζ inhibitor protein. The formation of crystals depends upon the presence of bound bacterial cardiolipin and phospholipid molecules; when they were removed, the complex failed to crystallize. The experiments open the way to an atomic structure of an F-ATPase complex. © 2015 The Authors.

Vacuolar H+-ATPase Protects Saccharomyces cerevisiae Cells against Ethanol-Induced Oxidative and Cell Wall Stresses.

Science.gov (United States)

Charoenbhakdi, Sirikarn; Dokpikul, Thanittra; Burphan, Thanawat; Techo, Todsapol; Auesukaree, Choowong

2016-05-15

During fermentation, increased ethanol concentration is a major stress for yeast cells. Vacuolar H(+)-ATPase (V-ATPase), which plays an important role in the maintenance of intracellular pH homeostasis through vacuolar acidification, has been shown to be required for tolerance to straight-chain alcohols, including ethanol. Since ethanol is known to increase membrane permeability to protons, which then promotes intracellular acidification, it is possible that the V-ATPase is required for recovery from alcohol-induced intracellular acidification. In this study, we show that the effects of straight-chain alcohols on membrane permeabilization and acidification of the cytosol and vacuole are strongly dependent on their lipophilicity. These findings suggest that the membrane-permeabilizing effect of straight-chain alcohols induces cytosolic and vacuolar acidification in a lipophilicity-dependent manner. Surprisingly, after ethanol challenge, the cytosolic pH in Δvma2 and Δvma3 mutants lacking V-ATPase activity was similar to that of the wild-type strain. It is therefore unlikely that the ethanol-sensitive phenotype of vma mutants resulted from severe cytosolic acidification. Interestingly, the vma mutants exposed to ethanol exhibited a delay in cell wall remodeling and a significant increase in intracellular reactive oxygen species (ROS). These findings suggest a role for V-ATPase in the regulation of the cell wall stress response and the prevention of endogenous oxidative stress in response to ethanol. The yeast Saccharomyces cerevisiae has been widely used in the alcoholic fermentation industry. Among the environmental stresses that yeast cells encounter during the process of alcoholic fermentation, ethanol is a major stress factor that inhibits yeast growth and viability, eventually leading to fermentation arrest. This study provides evidence for the molecular mechanisms of ethanol tolerance, which is a desirable characteristic for yeast strains used in alcoholic

Retinitis Pigmentosa Mutations in Bad Response to Refrigeration 2 (Brr2) Impair ATPase and Helicase Activity.

Science.gov (United States)

Ledoux, Sarah; Guthrie, Christine

2016-06-03

Brr2 is an RNA-dependent ATPase required to unwind the U4/U6 snRNA duplex during spliceosome assembly. Mutations within the ratchet helix of the Brr2 RNA binding channel result in a form of degenerative human blindness known as retinitis pigmentosa (RP). The biochemical consequences of these mutations on Brr2's RNA binding, helicase, and ATPase activity have not yet been characterized. Therefore, we identified the largest construct of Brr2 that is soluble in vitro, which truncates the first 247 amino acids of the N terminus (Δ247-Brr2), to characterize the effects of the RP mutations on Brr2 activity. The Δ247-Brr2 RP mutants exhibit a gradient of severity of weakened RNA binding, reduced helicase activity, and reduced ATPase activity compared with wild type Δ247-Brr2. The globular C-terminal Jab1/Mpn1-like domain of Prp8 increases the ability of Δ247-Brr2 to bind the U4/U6 snRNA duplex at high pH and increases Δ247-Brr2's RNA-dependent ATPase activity and the extent of RNA unwinding. However, this domain of Prp8 does not differentially affect the Δ247-Brr2 RP mutants compared with the wild type Δ247-Brr2. When stimulated by Prp8, wild type Δ247-Brr2 is able to unwind long stable duplexes in vitro, and even the RP mutants capable of binding RNA with tight affinity are incapable of fully unwinding short duplex RNAs. Our data suggest that the RP mutations within the ratchet helix impair Brr2 translocation through RNA helices. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

N-Terminal Pro-B-Type Natriuretic Peptide and Phonocardiography in Differentiating Innocent Cardiac Murmurs from Congenital Cardiac Anomalies in Asymptomatic Puppies

NARCIS (Netherlands)

Marinus, S M; Engelen, H.G.H.; Szatmári, V.

2017-01-01

Background: Differentiating innocent cardiac murmurs from murmurs caused by congenital cardiac anomalies can be challenging with auscultation alone in asymptomatic puppies. Hypothesis: Plasma N-terminal pro-B-type natriuretic peptide (NT-proBNP) concentrations and phonocardiograms recorded by an

Transcriptional regulators of Na, K-ATPase subunits

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Zhiqin eLi

2015-10-01

Full Text Available The Na,K-ATPase classically serves as an ion pump creating an electrochemical gradient across the plasma membrane that is essential for transepithelial transport, nutrient uptake and membrane potential. In addition, Na,K-ATPase also functions as a receptor, a signal transducer and a cell adhesion molecule. With such diverse roles, it is understandable that the Na,K-ATPase subunits, the catalytic alpha-subunit, the beta-subunit and the FXYD proteins, are controlled extensively during development and to accommodate physiological needs. The spatial and temporal expression of Na,K-ATPase is partially regulated at the transcriptional level. Numerous transcription factors, hormones, growth factors, lipids and extracellular stimuli modulate the transcription of the Na,K-ATPase subunits. Moreover, epigenetic mechanisms also contribute to the regulation of Na,K-ATPase expression. With the ever growing knowledge about diseases associated with the malfunction of Na,K-ATPase, this review aims at summarizing the best-characterized transcription regulators that modulate Na,K-ATPase subunit levels. As abnormal expression of Na,K-ATPase subunits have been observed in many carcinoma, we will also discuss transcription factors that are associated with epithelial-to-mesenchymal transition, a crucial step in the progression of many tumors to malignant disease.

Molecular dissection of the C-terminal regulatory domain of the plant plasma membrane H+-ATPase AHA2: Mapping of residues that when altered give rise to an activated enzyme

DEFF Research Database (Denmark)

Axelsen, K.B.; Venema, K.; Jah, T.

1999-01-01

in an extension of the C-terminus unique to plant H+-ATPases, Alteration of residues in both regions led to increased binding of yeast 14-3-3 protein to the plasma membrane of transformed cells. Taken together, our data suggest that modification of residues in two regions of the C-terminal regulatory domain......The plasma membrane H+-ATPase is a proton pump belonging to the P-type ATPase superfamily and is important for nutrient acquisition in plants, The H+-ATPase is controlled by an autoinhibitory C-terminal regulatory domain and is activated by 14-3-3 proteins which bind to this part of the enzyme......+-ATPase. The enzymes were characterized by their ability to promote growth in acidic conditions and to promote H+ extrusion from intact cells, both of which are measures of plasma membrane H+-ATPase activity, and were also characterized with respect to kinetic properties such as affinity for H+ and ATP. Residues...

Distribution of fast myosin heavy chain-based muscle fibres in the gluteus medius of untrained horses: mismatch between antigenic and ATPase determinants

Science.gov (United States)

LINNANE, LINDA; SERRANO, A. L.; RIVERO, J. L. L.

1999-01-01

The distribution of muscle fibres classified on the basis of their content of different myosin heavy chain (MHC) isoforms was analysed in muscle biopsies from the gluteus medius of adult untrained horses by correlating immunohistochemistry with specific anti-MHC monoclonal antibodies and standard myofibrillar ATPase (mATPase) histochemistry. Percutaneous needle biopsies were taken at 3 depths (20, 40 and 60 mm) from 4 4-y-old Andalusian stallions. The percentage of ‘pure’ I MHC fibres increased whereas that for pure IIX MHC fibres decreased from the most superficial to the deepest sampling site. Within the fast fibres, types IIA and IIAX MHC-classified fibres were proportionately more abundant in the deepest sampling site than in the superficial region of the muscle. The immunohistochemical and histochemical characterisation of a large number of single fibres (n=1375) was compared and correlated on a fibre-to-fibre basis. The results showed that 40% of the fibres analysed were pure type I (expressing only MHC-I); they showed correct matching between their antigenic and mATPase determinants. In contrast, within the fast fibres, a considerable proportion of fibres were found showing a mismatch between their immunohistochemical and mATPase profiles. The most common mismatched fibre phenotypes comprised fibres displaying coexpression of both fast MHCs when analysed by immunocytochemistry, but showing an mATPase profile similar to typical IIX fibres (moderate mATPase reaction after preincubation at pH 4.4). Considered altogether, the total mismatched fibres represented only 4.2% of the whole fast fibre population in the superficial region of the muscle, but their proportion increased to 15.6% and 38.4% in the middle and deep regions, respectively, of gluteus medius. It is concluded that a considerable number of hybrid fast MHC IIAX fibres are present in the gluteus medius of untrained horses, suggesting that equine type II fibres have probably been misclassified in

Inositol phosphates influence the membrane bound Ca2+/Mg2+ stimulated ATPase from human erythrocyte membranes

International Nuclear Information System (INIS)

Kester, M.; Ekholm, J.; Kumar, R.; Hanahan, D.J.

1986-01-01

The modulation by exogenous inositol phosphates of the membrane Ca 2+ /Mg 2+ ATPase from saponin/EGTA lysed human erythrocytes was determined in a buffer (pH 7.6) containing histidine, 80 mM, MgCl 2 , 3.3 mM, NaCl, 74 mM, KCl, 30 mM, Na 2 ATP, 2.3 mM, ouabain, 0.83 mM, with variable amounts of CaCl 2 and EGTA. The ATPase assay was linear with time at 44 0 C. The inositol phosphates were commercially obtained and were also prepared from 32 P labeled rabbit platelet inositol phospholipids. Inositol triphosphate (IP 3 ) elevated the Ca 2+ /Mg 2+ ATPase activity over basal levels in a dose, time, and calcium dependent manner and were increased up to 85% of control values. Activities for the Na + /K + -ATPase and a Mg 2+ ATPase were not effected by IP 3 . Ca 2+ /Mg 2+ APTase activity with IP 2 or IP 3 could be synergistically elevated with calmodulin addition. The activation of the ATPase with IP 3 was calcium dependent in a range from .001 to .02 mM. The apparent Km and Vmax values were determined for IP 3 stimulated Ca 2+ /Mg 2+ ATPase

Organophosphate inhibition of avian salt gland Na, K-ATPase activity

Science.gov (United States)

Eastin, W.C.; Fleming, W.J.; Murray, H.C.

1982-01-01

1. Adult black ducks (Anas rubripes) were given freshwater or saltwater (1.5% NaCl) for 11 days and half of each group was also given an organophosphate (17 p.p.m. fenthion) in the diet on days 6–11.2. After 11 days, ducks drinking saltrwater had lost more weight and had higher plasma Na and uric acid concentration and osmolalities than birds drinking freshwater.3. Saltwater treatment stimulated the salt gland to increased weight and Na, K-ATPase activity.4. Fenthion generally reduced plasma and brain cholinesterase activity and depressed cholinesterase and Na, K-ATPase activities in salt glands of birds drinking saltwater.

Oxidative stress (glutathionylation and Na,K-ATPase activity in rat skeletal muscle.

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Carsten Juel

Full Text Available Changes in ion distribution across skeletal muscle membranes during muscle activity affect excitability and may impair force development. These changes are counteracted by the Na,K-ATPase. Regulation of the Na,K-ATPase is therefore important for skeletal muscle function. The present study investigated the presence of oxidative stress (glutathionylation on the Na,K-ATPase in rat skeletal muscle membranes.Immunoprecipitation with an anti-glutathione antibody and subsequent immunodetection of Na,K-ATPase protein subunits demonstrated 9.0±1.3% and 4.1±1.0% glutathionylation of the α isoforms in oxidative and glycolytic skeletal muscle, respectively. In oxidative muscle, 20.0±6.1% of the β1 units were glutathionylated, whereas 14.8±2.8% of the β2-subunits appear to be glutathionylated in glycolytic muscle. Treatment with the reducing agent dithiothreitol (DTT, 1 mM increased the in vitro maximal Na,K-ATPase activity by 19% (P<0.05 in membranes from glycolytic muscle. Oxidized glutathione (GSSG, 0-10 mM increased the in vitro glutathionylation level detected with antibodies, and decreased the in vitro maximal Na,K-ATPase activity in a dose-dependent manner, and with a larger effect in oxidative compared to glycolytic skeletal muscle.This study demonstrates the existence of basal glutathionylation of both the α and the β units of rat skeletal muscle Na,K-ATPase. In addition, the study suggests a negative correlation between glutathionylation levels and maximal Na,K-ATPase activity.Glutathionylation likely contributes to the complex regulation of Na,K-ATPase function in skeletal muscle. Especially, glutathionylation induced by oxidative stress may have a role in Na,K-ATPase regulation during prolonged muscle activity.

Effect of near-UV light on Na-K-ATPase of the rat lens

Energy Technology Data Exchange (ETDEWEB)

Torriglia, A.; Zigman, S.

1988-06-01

The influence of in vitro near-UV radiation exposure on the physical state of the rat lens and on its membrane-bound Na-K-ATPase activity was investigated. Lens swelling was correlated to the appearance of opacities and the inactivation of the enzyme. The results show a significant decrease in the Na-K-ATPase activity which may be an early change leading to osmotic type cataracts. The dose-effect curves obtained for cortical and epithelial enzymes were different. Since the data do not follow a mono-exponential function, the existence of two forms of Na-K-ATPase in the lens is discussed.

The role of Na+/K+-ATPase during chick skeletal myogenesis.

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Taissa Neustadt Oliveira

Full Text Available The formation of a vertebrate skeletal muscle fiber involves a series of sequential and interdependent events that occurs during embryogenesis. One of these events is myoblast fusion which has been widely studied, yet not completely understood. It was previously shown that during myoblast fusion there is an increase in the expression of Na+/K+-ATPase. This fact prompted us to search for a role of the enzyme during chick in vitro skeletal myogenesis. Chick myogenic cells were treated with the Na+/K+-ATPase inhibitor ouabain in four different concentrations (0.01-10 μM and analyzed. Our results show that 0.01, 0.1 and 1 μM ouabain did not induce changes in cell viability, whereas 10 μM induced a 45% decrease. We also observed a reduction in the number and thickness of multinucleated myotubes and a decrease in the number of myoblasts after 10 μM ouabain treatment. We tested the involvement of MEK-ERK and p38 signaling pathways in the ouabain-induced effects during myogenesis, since both pathways have been associated with Na+/K+-ATPase. The MEK-ERK inhibitor U0126 alone did not alter cell viability and did not change ouabain effect. The p38 inhibitor SB202190 alone or together with 10 μM ouabain did not alter cell viability. Our results show that the 10 μM ouabain effects in myofiber formation do not involve the MEK-ERK or the p38 signaling pathways, and therefore are probably related to the pump activity function of the Na+/K+-ATPase.

Structural and Biochemical Characterization of Spa47 Provides Mechanistic Insight into Type III Secretion System ATPase Activation and Shigella Virulence Regulation*

Science.gov (United States)

Burgess, Jamie L.; Burgess, R. Alan; Morales, Yalemi; Bouvang, Jenna M.; Johnson, Sean J.; Dickenson, Nicholas E.

2016-01-01

Like many Gram-negative pathogens, Shigella rely on a complex type III secretion system (T3SS) to inject effector proteins into host cells, take over host functions, and ultimately establish infection. Despite these critical roles, the energetics and regulatory mechanisms controlling the T3SS and pathogen virulence remain largely unclear. In this study, we present a series of high resolution crystal structures of Spa47 and use the structures to model an activated Spa47 oligomer, finding that ATP hydrolysis may be supported by specific side chain contributions from adjacent protomers within the complex. Follow-up mutagenesis experiments targeting the predicted active site residues validate the oligomeric model and determined that each of the tested residues are essential for Spa47 ATPase activity, although they are not directly responsible for stable oligomer formation. Although N-terminal domain truncation was necessary for crystal formation, it resulted in strictly monomeric Spa47 that is unable to hydrolyze ATP, despite maintaining the canonical ATPase core structure and active site residues. Coupled with studies of ATPase inactive full-length Spa47 point mutants, we find that Spa47 oligomerization and ATP hydrolysis are needed for complete T3SS apparatus formation, a proper translocator secretion profile, and Shigella virulence. This work represents the first structure-function characterization of Spa47, uniquely complementing the multitude of included Shigella T3SS phenotype assays and providing a more complete understanding of T3SS ATPase-mediated pathogen virulence. Additionally, these findings provide a strong platform for follow-up studies evaluating regulation of Spa47 oligomerization in vivo as a much needed means of treating and perhaps preventing shigellosis. PMID:27770024

Effect of hindlimb unweighting on single soleus fiber maximal shortening velocity and ATPase activity

Science.gov (United States)

Mcdonald, K. S.; Fitts, R. H.

1993-01-01

The effect of hindlimb unweighting (HU) for 1 to 3 wks on the shortening velocity of a soleus fiber, its ATPase content, and the relative contents of the slow and fast myosin was investigated by measuring fiber force, V(0), ATPase activity, and myosin content in SDS protein profiles of a single rat soleus fiber suspended between a motor arm and a transducer. It was found that HU induces a progressive increase in fiber V(0) that is likely caused, at least in part, by an increase in the fiber's myofibrillar ATPase activity. The HU-induced increases in V(0) and ATPase were associated with the presence of a greater percentage of fast type IIa fibers. However, a large population of fibers after 1, 2, and 3 wks of HU showed increases in V(0) and ATPase but displayed the same myosin protein profile on SDS gels as control fibers.

Environmental and Genetic Factors Regulating Localization of the Plant Plasma Membrane H+-ATPase1[OPEN

Science.gov (United States)

Tan, Li Xuan; Bushey, Daniel B.; Swanson, Sarah J.

2018-01-01

A P-type H+-ATPase is the primary transporter that converts ATP to electrochemical energy at the plasma membrane of higher plants. Its product, the proton-motive force, is composed of an electrical potential and a pH gradient. Many studies have demonstrated that this proton-motive force not only drives the secondary transporters required for nutrient uptake, but also plays a direct role in regulating cell expansion. Here, we have generated a transgenic Arabidopsis (Arabidopsis thaliana) plant expressing H+-ATPase isoform 2 (AHA2) that is translationally fused with a fluorescent protein and examined its cellular localization by live-cell microscopy. Using a 3D imaging approach with seedlings grown for various times under a variety of light intensities, we demonstrate that AHA2 localization at the plasma membrane of root cells requires light. In dim light conditions, AHA2 is found in intracellular compartments, in addition to the plasma membrane. This localization profile was age-dependent and specific to cell types found in the transition zone located between the meristem and elongation zones. The accumulation of AHA2 in intracellular compartments is consistent with reduced H+ secretion near the transition zone and the suppression of root growth. By examining AHA2 localization in a knockout mutant of a receptor protein kinase, FERONIA, we found that the intracellular accumulation of AHA2 in the transition zone is dependent on a functional FERONIA-dependent inhibitory response in root elongation. Overall, this study provides a molecular underpinning for understanding the genetic, environmental, and developmental factors influencing root growth via localization of the plasma membrane H+-ATPase. PMID:29042459

Expression of a constitutively activated plasma membrane H+-ATPase in Nicotiana tabacum BY-2 cells results in cell expansion.

Science.gov (United States)

Niczyj, Marta; Champagne, Antoine; Alam, Iftekhar; Nader, Joseph; Boutry, Marc

2016-11-01

Increased acidification of the external medium by an activated H + -ATPase results in cell expansion, in the absence of upstream activating signaling. The plasma membrane H + -ATPase couples ATP hydrolysis with proton transport outside the cell, and thus creates an electrochemical gradient, which energizes secondary transporters. According to the acid growth theory, this enzyme is also proposed to play a major role in cell expansion, by acidifying the external medium and so activating enzymes that are involved in cell wall-loosening. However, this theory is still debated. To challenge it, we made use of a plasma membrane H + -ATPase isoform from Nicotiana plumbaginifolia truncated from its C-terminal auto-inhibitory domain (ΔCPMA4), and thus constitutively activated. This protein was expressed in Nicotiana tabacum BY-2 suspension cells using a heat shock inducible promoter. The characterization of several independent transgenic lines showed that the expression of activated ΔCPMA4 resulted in a reduced external pH by 0.3-1.2 units, as well as in an increased H + -ATPase activity by 77-155 % (ATP hydrolysis), or 70-306 % (proton pumping) of isolated plasma membranes. In addition, ΔCPMA4-expressing cells were 17-57 % larger than the wild-type cells and displayed abnormal shapes. A proteomic comparison of plasma membranes isolated from ΔCPMA4-expressing and wild-type cells revealed the altered abundance of several proteins involved in cell wall synthesis, transport, and signal transduction. In conclusion, the data obtained in this work showed that H + -ATPase activation is sufficient to induce cell expansion and identified possible actors which intervene in this process.

FXYD5: Na+/ K+-ATPase Regulator in Health and Disease.

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Irina eLubarski Gotliv

2016-03-01

Full Text Available FXYD5 (Dysadherin, RIC is a single span type I membrane protein that plays multiple roles in regulation of cellular functions. It is expressed in a variety of epithelial tissues and acts as an auxiliary subunit of the Na+/ K+-ATPase. During the past decade, a correlation between enhanced expression of FXYD5 and tumor progression has been established for various tumor types. In this review, current knowledge on FXYD5 is discussed, including experimental data on the functional effects of FXYD5 on the Na+/ K+-ATPase. FXYD5 modulates cellular junctions, influences chemokine production, and affects cell adhesion. The accumulated data may provide a basis for understanding the molecular mechanisms underlying FXYD5 mediated phenotypes.

Mechanism of Na,K-ATPase decline during sheep red cell maturation

International Nuclear Information System (INIS)

Grafova, E.; Blostein, R.

1987-01-01

Na,K-ATPase of immature and mature sheep red cells of both the high-K + and low-K + genotypes as well as cells of both types matured in vitro was detected using polyclonal antiserum to sheep kidney Na,K-ATPase. Following SDS-PAGE and immunoblotting, the major reactive component was the ∼ 100 kDa catalytic α subunit. A less prominent band migrating as a sharper, lower molecular weight (50 kDa) component than the kidney Na,K-ATPase β subunit is apparent in reticulocytes but not mature cells. Membranes from both genotypes showed identical immunologically reactive peptides, except for the lower intensity of the α subunit in the mature cells of the low- compared to high-K + sheep. Following culture of both types, moderate reduction in reactivity was apparent. Immunologically reactive α subunit as well as the 50 kDa species were detected in membranous material shed into the culture medium. This material was functionally inactive (lack of both [ 3 H] ouabain binding and Na + -dependent phosphorylation of Na,K-ATPase). The existence in reticulocytes of an intracellular pool of ouabain binding sites is evidenced in appearance of extra sites following rapid ATP depletion and also after addition of chloroquine. Taken together, these findings are consistent with a maturation-associated decrease of sodium pumps by a process of membrane recycling, processing and, to some extent, exocytosis

Hypertension is a conditional factor for the development of cardiac hypertrophy in type 2 diabetic mice.

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Marc van Bilsen

Full Text Available BACKGROUND: Type 2 diabetes is frequently associated with co-morbidities, including hypertension. Here we investigated if hypertension is a critical factor in myocardial remodeling and the development of cardiac dysfunction in type 2 diabetic db/db mice. METHODS: Thereto, 14-wks-old male db/db mice and non-diabetic db/+ mice received vehicle or angiotensin II (AngII for 4 wks to induce mild hypertension (n = 9-10 per group. Left ventricular (LV function was assessed by serial echocardiography and during a dobutamine stress test. LV tissue was subjected to molecular and (immunohistochemical analysis to assess effects on hypertrophy, fibrosis and inflammation. RESULTS: Vehicle-treated diabetic mice neither displayed marked myocardial structural remodeling nor cardiac dysfunction. AngII-treatment did not affect body weight and fasting glucose levels, and induced a comparable increase in blood pressure in diabetic and control mice. Nonetheless, AngII-induced LV hypertrophy was significantly more pronounced in diabetic than in control mice as assessed by LV mass (increase +51% and +34%, respectively, p<0.01 and cardiomyocyte size (+53% and +31%, p<0.001. This was associated with enhanced LV mRNA expression of markers of hypertrophy and fibrosis and reduced activation of AMP-activated protein kinase (AMPK, while accumulation of Advanced Glycation End products (AGEs and the expression levels of markers of inflammation were not altered. Moreover, AngII-treatment reduced LV fractional shortening and contractility in diabetic mice, but not in control mice. CONCLUSIONS: Collectively, the present findings indicate that type 2 diabetes in its early stage is not yet associated with adverse cardiac structural changes, but already renders the heart more susceptible to hypertension-induced hypertrophic remodeling.

Amino Acid Availability Modulates Vacuolar H+-ATPase Assembly*

Science.gov (United States)

Stransky, Laura A.; Forgac, Michael

2015-01-01

The vacuolar H+-ATPase (V-ATPase) is an ATP-dependent proton pump composed of a peripheral ATPase domain (V1) and a membrane-integral proton-translocating domain (V0) and is involved in many normal and disease processes. An important mechanism of regulating V-ATPase activity is reversible assembly of the V1 and V0 domains. Increased assembly in mammalian cells occurs under various conditions and has been shown to involve PI3K. The V-ATPase is necessary for amino acid-induced activation of mechanistic target of rapamycin complex 1 (mTORC1), which is important in controlling cell growth in response to nutrient availability and growth signals. The V-ATPase undergoes amino acid-dependent interactions with the Ragulator complex, which is involved in recruitment of mTORC1 to the lysosomal membrane during amino acid sensing. We hypothesized that changes in the V-ATPase/Ragulator interaction might involve amino acid-dependent changes in V-ATPase assembly. To test this, we measured V-ATPase assembly by cell fractionation in HEK293T cells treated with and without amino acids. V-ATPase assembly increases upon amino acid starvation, and this effect is reversed upon readdition of amino acids. Lysosomes from amino acid-starved cells possess greater V-ATPase-dependent proton transport, indicating that assembled pumps are catalytically active. Amino acid-dependent changes in both V-ATPase assembly and activity are independent of PI3K and mTORC1 activity, indicating the involvement of signaling pathways distinct from those implicated previously in controlling assembly. By contrast, lysosomal neutralization blocks the amino acid-dependent change in assembly and reactivation of mTORC1 after amino acid starvation. These results identify an important new stimulus for controlling V-ATPase assembly. PMID:26378229

A STUDY ON ASYMPTOMATIC CARDIAC CHANGES IN TYPE 2 DIABETES PATIENTS

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Ramchandra Rao

2015-06-01

Full Text Available INTRODUCTION: Majority of the time the patient of Diabetes presents with complications like Myocardial infarction, heart failure, being end stages of cardiovascular 2 disease associated with other macro and microvascular complications. If patients are screened at an early stage of Diabetes before the onset of symptoms the cardiovascular complications can be delayed and mortality can be reduced . There are only few studies in India done to screen asymptomatic diabetic individuals for cardiovascular compli cations. This study is done in view of screening the asymptomatic individuals in our area to prevent complications. With the available infrastructure, ECG, 2D ECHO were done in 50 patients who met inclusion, exclusion criterias, the changes were noted and the cardiac status was evaluated. AIM AND OBJECTIVES : “To study the asymptomatic cardiac changes in type2 Diabetes patients”. To observe the ECG changes in patients of type2 Diabetes without any symptoms of cardiac disease. To evaluate the ECG changes along with 2DECHO findings in asymptomatic cardiac patients of type 2 Diabetes. To study the correlation between these two investigations and evaluate the cardiac status of the individual. CONCLUSIONS : M ost of the patients in study group belong to 5 th decade , Males with Diabetes were almost double that of females , Most of the patients had duration of Diabetes as 5yrs , Less than half of patients had family history of Diabetes , Half of the group had alcohol, smoking habits , More than half of patients were overweight , Only 20% had good control of Diabetes , Total cholesterol is above normal in almost all of the patients, LDL is elevated in half of the patients, triglycerides in most of them, there is significant dyslipidemia in patients of Diabetes , Only 6 had normal ECG. Rest of them have LVH, ischemia , 18 patients had changes in echo including LVD, regional and global hypokinesias , t hus the present study shows patients with type 2

Two classes of ouabain binding sites in ferret heart and two forms of Na+-K+-ATPase

Energy Technology Data Exchange (ETDEWEB)

Ng, Y.C.; Akera, T.

1987-05-01

In partially purified Na+-K+-adenosinetriphosphatase (ATPase) obtained from ferret heart, ouabain produced a monophasic inhibition curve; however, the curve spanned over 5 logarithmic units, indicating the presence of more than one classes of enzyme. (/sup 3/H)ouabain binding studies revealed high-and low-affinity binding sites in approximately equal abundance, with apparent dissociation constants of 10 and 230 nM, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of phosphoenzyme formed from (gamma-/sup 32/P)ATP showed two distinct K+-sensitive bands of approximately 100,000 molecular weight. Phosphoenzyme formation from the high-molecular-weight alpha(+) form was selectively inhibited by N-ethylmaleimide. Ouabain caused a 50% inhibition of phosphorylation of the alpha(+) form at 40 nM and the lower-molecular-weight alpha form at 300 nM. In papillary muscle preparations, 1-30 nM ouabain produced a modest positive inotropic effect that reached an apparent plateau at 30 nM. Further increases in ouabain concentrations, however, produced additional and prominent inotropic effects at 0.1-10 microM. These results indicate for the first time in cardiac muscle that the high- and low-affinity ouabain binding sites are associated with the alpha(+) and alpha forms of the Na+-K+-ATPase, respectively, and that binding of ouabain to either of these sites causes enzyme inhibition and the positive inotropic effect.

Spermine modulates fungal morphogenesis and activates plasma membrane H+-ATPase during yeast to hyphae transition

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Antônio Jesus Dorighetto Cogo

2018-02-01

Full Text Available Polyamines play a regulatory role in eukaryotic cell growth and morphogenesis. Despite many molecular advances, the underlying mechanism of action remains unclear. Here, we investigate a mechanism by which spermine affects the morphogenesis of a dimorphic fungal model of emerging relevance in plant interactions, Yarrowia lipolytica, through the recruitment of a phytohormone-like pathway involving activation of the plasma membrane P-type H+-ATPase. Morphological transition was followed microscopically, and the H+-ATPase activity was analyzed in isolated membrane vesicles. Proton flux and acidification were directly probed at living cell surfaces by a non-invasive selective ion electrode technique. Spermine and indol-3-acetic acid (IAA induced the yeast-hypha transition, influencing the colony architecture. Spermine induced H+-ATPase activity and H+ efflux in living cells correlating with yeast-hypha dynamics. Pharmacological inhibition of spermine and IAA pathways prevented the physio-morphological responses, and indicated that spermine could act upstream of the IAA pathway. This study provides the first compelling evidence on the fungal morphogenesis and colony development as modulated by a spermine-induced acid growth mechanism analogous to that previously postulated for the multicellular growth regulation of plants.

"Just-In-Time" Simulation Training Using 3-D Printed Cardiac Models After Congenital Cardiac Surgery.

Science.gov (United States)

Olivieri, Laura J; Su, Lillian; Hynes, Conor F; Krieger, Axel; Alfares, Fahad A; Ramakrishnan, Karthik; Zurakowski, David; Marshall, M Blair; Kim, Peter C W; Jonas, Richard A; Nath, Dilip S

2016-03-01

High-fidelity simulation using patient-specific three-dimensional (3D) models may be effective in facilitating pediatric cardiac intensive care unit (PCICU) provider training for clinical management of congenital cardiac surgery patients. The 3D-printed heart models were rendered from preoperative cross-sectional cardiac imaging for 10 patients undergoing congenital cardiac surgery. Immediately following surgical repair, a congenital cardiac surgeon and an intensive care physician conducted a simulation training session regarding postoperative care utilizing the patient-specific 3D model for the PCICU team. After the simulation, Likert-type 0 to 10 scale questionnaire assessed participant perception of impact of the training session. Seventy clinicians participated in training sessions, including 22 physicians, 38 nurses, and 10 ancillary care providers. Average response to whether 3D models were more helpful than standard hand off was 8.4 of 10. Questions regarding enhancement of understanding and clinical ability received average responses of 9.0 or greater, and 90% of participants scored 8 of 10 or higher. Nurses scored significantly higher than other clinicians on self-reported familiarity with the surgery (7.1 vs. 5.8; P = .04), clinical management ability (8.6 vs. 7.7; P = .02), and ability enhancement (9.5 vs. 8.7; P = .02). Compared to physicians, nurses and ancillary providers were more likely to consider 3D models more helpful than standard hand off (8.7 vs. 7.7; P = .05). Higher case complexity predicted greater enhancement of understanding of surgery (P = .04). The 3D heart models can be used to enhance congenital cardiac critical care via simulation training of multidisciplinary intensive care teams. Benefit may be dependent on provider type and case complexity. © The Author(s) 2016.

A high-yield co-expression system for the purification of an intact drs2p-cdc50p lipid flippase complex, critically dependent on and stabilized by phosphatidylinositol-4-phosphate

DEFF Research Database (Denmark)

Azouaoui, Hassina; Montigny, Cédric; Ash, Miriam-Rose

2014-01-01

P-type ATPases from the P4 subfamily (P4-ATPases) are energy-dependent transporters, which are thought to establish lipid asymmetry in eukaryotic cell membranes. Together with their Cdc50 accessory subunits, P4-ATPases couple ATP hydrolysis to lipid transport from the exoplasmic to the cytoplasmic...... leaflet of plasma membranes, late Golgi membranes, and endosomes. To gain insights into the structure and function of these important membrane pumps, robust protocols for expression and purification are required. In this report, we present a procedure for high-yield co-expression of a yeast flippase......, the Drs2p-Cdc50p complex. After recovery of yeast membranes expressing both proteins, efficient purification was achieved in a single step by affinity chromatography on streptavidin beads, yielding ∼1-2 mg purified Drs2p-Cdc50p complex per liter of culture. Importantly, the procedure enabled us to recover...

The Kdp-ATPase system and its regulation

Indian Academy of Sciences (India)

2007-03-15

Mar 15, 2007 ... K+, the dominant intracellular cation, is required for various physiological processes like turgor homeostasis, pH regulation etc. Bacterial cells have evolved many diverse K+ transporters to maintain the desired concentration of internal K+. In E. coli, the KdpATPase (comprising of the KdpFABC complex), ...

Cardiac Function in 7-8-Year-Old Offspring of Women with Type 1 Diabetes

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Maarten Rijpert

2011-01-01

Full Text Available Offspring of type 1 diabetic mothers (ODMs are at risk of short-term and long-term complications, such as neonatal macrosomia (birth weight >90th percentile, hypertrophic cardiomyopathy, and cardiovascular morbidity in later life. However, no studies have been performed regarding cardiac outcome. In this study, we investigated cardiac dimensions and function in 30 ODMs at 7-8 years of age in relation to neonatal macrosomia and maternal glycemic control during pregnancy and compared these with those in a control group of 30 children of nondiabetic women. We found that cardiac dimensions and systolic and diastolic function parameters in ODMs were comparable with those in controls. Neonatal macrosomia and poorer maternal glycemic control during pregnancy were not related to worse cardiac outcome in ODM. We conclude that cardiac function at 7-8 years of age in offspring of women with type 1 diabetes is reassuring and comparable with that in controls.

Structural and Biochemical Characterization of Spa47 Provides Mechanistic Insight into Type III Secretion System ATPase Activation and Shigella Virulence Regulation.

Science.gov (United States)

Burgess, Jamie L; Burgess, R Alan; Morales, Yalemi; Bouvang, Jenna M; Johnson, Sean J; Dickenson, Nicholas E

2016-12-09

Like many Gram-negative pathogens, Shigella rely on a complex type III secretion system (T3SS) to inject effector proteins into host cells, take over host functions, and ultimately establish infection. Despite these critical roles, the energetics and regulatory mechanisms controlling the T3SS and pathogen virulence remain largely unclear. In this study, we present a series of high resolution crystal structures of Spa47 and use the structures to model an activated Spa47 oligomer, finding that ATP hydrolysis may be supported by specific side chain contributions from adjacent protomers within the complex. Follow-up mutagenesis experiments targeting the predicted active site residues validate the oligomeric model and determined that each of the tested residues are essential for Spa47 ATPase activity, although they are not directly responsible for stable oligomer formation. Although N-terminal domain truncation was necessary for crystal formation, it resulted in strictly monomeric Spa47 that is unable to hydrolyze ATP, despite maintaining the canonical ATPase core structure and active site residues. Coupled with studies of ATPase inactive full-length Spa47 point mutants, we find that Spa47 oligomerization and ATP hydrolysis are needed for complete T3SS apparatus formation, a proper translocator secretion profile, and Shigella virulence. This work represents the first structure-function characterization of Spa47, uniquely complementing the multitude of included Shigella T3SS phenotype assays and providing a more complete understanding of T3SS ATPase-mediated pathogen virulence. Additionally, these findings provide a strong platform for follow-up studies evaluating regulation of Spa47 oligomerization in vivo as a much needed means of treating and perhaps preventing shigellosis. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

Structural studies of the vacuolar membrane ATPase from Neurospora crassa and comparison with the tonoplast membrane ATPase and Zea mays

International Nuclear Information System (INIS)

Bowman, E.J.; Mandala, S.; Taiz, L.; Bowman, B.J.

1986-01-01

The H + translocating ATPase located on vacuolar membranes of Neurospora crassa was partially purified by solubilization in two detergents, Triton X-100 and N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate, followed by centrifugation on sucrose density gradients. Two polypeptides of M/sub r/ ≅ 70,000 and ≅ 62,000 consistently migrated with activity, along with several minor bands of lower molecular weight. Radioactively labeled inhibitors of ATPase activity, N-[ 14 C]ethylmaleimide and 7-chloro-4-nitro[ 14 C]benzo-2-oxa-1,3-diazole, labeled the M/sub r/ ≅ 70,000 polypeptide; this labeling was reduced in the presence of ATP. N,N'-[ 14 C]dicyclohexylcarbodiimide labeled a polypeptide of M/sub r/ ≅ 15,000. Estimation of the functional size of the vacuolar membrane ATPase by radiation inactivation gave a value of M/sub r/ 5.2 x 10 5 , 10-15% larger than the mitochondrial ATPase. The Neurospora vacuolar ATPase showed no crossreactivity with antiserum to plasma membrane or mitochrondrial ATPase but stongly crossreacted with antiserum against a polypeptide of M/sub r/ ≅ 70,000 associated with the tonoplast ATPase of corn coleoptiles. These results suggest that fungal and plant vacuolar ATPases may be large multisubunit complexes, somewhat similar to, but immunologically distinct from, known F 0 F 1 ATPases

The effect of near-UV light on Na-K-ATPase of the rat lens

International Nuclear Information System (INIS)

Torriglia, A.; Zigman, S.

1988-01-01

The influence of in vitro near-UV radiation exposure on the physical state of the rat lens and on its membrane-bound Na-K-ATPase activity was investigated. Lens swelling was correlated to the appearance of opacities and the inactivation of the enzyme. The results show a significant decrease in the Na-K-ATPase activity which may be an early change leading to osmotic type cataracts. The dose-effect curves obtained for cortical and epithelial enzymes were different. Since the data do not follow a mono-exponential function, the existence of two forms of Na-K-ATPase in the lens is discussed. (author)

Plant-derived cardiac glycosides: Role in heart ailments and cancer management.

Science.gov (United States)

Patel, Seema

2016-12-01

Cardiac glycosides, the cardiotonic steroids such as digitalis have been in use as heart ailment remedy since ages. They manipulate the renin-angiotensin axis to improve cardiac output. However; their safety and efficacy have come under scrutiny in recent times, as poisoning and accidental mortalities have been observed. In order to better understand and exploit them as cardiac ionotropes, studies are being pursued using different cardiac glycosides such as digitoxin, digoxin, ouabain, oleandrin etc. Several cardiac glycosides as peruvoside have shown promise in cancer control, especially ovary cancer and leukemia. Functional variability of these glycosides has revealed that not all cardiac glycosides are alike. Apart from their specific affinity to sodium-potassium ATPase, their therapeutic dosage and behavior in poly-morbidity conditions needs to be considered. This review presents a concise account of the key findings in recent years with adequate elaboration of the mechanisms. This compilation is expected to contribute towards management of cardiac, cancer, even viral ailments. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Hormonal regulation of Na+-K+-ATPase in cultured epithelial cells

International Nuclear Information System (INIS)

Johnson, J.P.; Jones, D.; Wiesmann, W.P.

1986-01-01

Aldosterone and insulin stimulate Na + transport through mechanisms involving protein synthesis. Na + -K + -ATPase has been implicated in the action of both hormones. The authors examined the effect of aldosterone and insulin on Na + -K + -ATPase in epithelial cells in culture derived from toad urinary bladder (TB6C) and toad kidney (A6). Aldosterone, but not insulin, increases short-circuit current (I/sub sc/) in TB6C cells. Aldosterone increases Na + -K + -[ 32 P]ATPase activity after 18 h of incubation, but no effect can be seen at 3 and 6 h. Amiloride, which inhibits aldosterone-induced increases in I/sub sc/, has no effect on either basal or aldosterone stimulated enzyme activity. Both aldosterone and insulin increase I/sub sc/ in A6 cells and when added together are synergistic. Aldosterone stimulates enzyme activity in A6 cells, but insulin alone has no effect. However, aldosterone and insulin together stimulate enzyme activity more than aldosterone alone. It appears that stimulation of Na + -K + -ATPase activity is involved in aldosterone action in both cell lines but does not appear to be due to increased Na + entry, since enhanced enzyme activity is not inhibited by amiloride. In contrast, insulin alone has no direct effect on Na + -K + -ATPase, although the increased enzyme activity following both agents in combination may explain their synergism on I/sub sc/

A Mighty “Protein Extractor” of the Cell: Structure and Function of the p97/CDC48 ATPase

Directory of Open Access Journals (Sweden)

Yihong Ye

2017-06-01

Full Text Available p97/VCP (known as Cdc48 in S. cerevisiae or TER94 in Drosophila is one of the most abundant cytosolic ATPases. It is highly conserved from archaebacteria to eukaryotes. In conjunction with a large number of cofactors and adaptors, it couples ATP hydrolysis to segregation of polypeptides from immobile cellular structures such as protein assemblies, membranes, ribosome, and chromatin. This often results in proteasomal degradation of extracted polypeptides. Given the diversity of p97 substrates, this “segregase” activity has profound influence on cellular physiology ranging from protein homeostasis to DNA lesion sensing, and mutations in p97 have been linked to several human diseases. Here we summarize our current understanding of the structure and function of this important cellular machinery and discuss the relevant clinical implications.

Altered expression and insulin-induced trafficking of Na+-K+-ATPase in rat skeletal muscle

DEFF Research Database (Denmark)

Galuska, Dana; Kotova, Olga; Barres, Romain

2009-01-01

Skeletal muscle Na(+)-K(+)-ATPase plays a central role in the clearance of K(+) from the extracellular fluid, therefore maintaining blood [K(+)]. Na(+)-K(+)-ATPase activity in peripheral tissue is impaired in insulin resistant states. We determined effects of high-fat diet (HFD) and exercise......(+)-K(+)-ATPase activity after 4 wk of HFD. Exercise training restored alpha(1)-, alpha(2)-, and beta(1)-subunit expression and Na(+)-K(+)-ATPase activity to control levels and reduced beta(2)-subunit expression 2.2-fold (P ... phospholemman. Phospholemman mRNA and protein expression were increased after HFD and restored to control levels after ET. Insulin-stimulated translocation of the alpha(2)-subunit to plasma membrane was impaired by HFD, whereas alpha(1)-subunit translocation remained unchanged. Alterations in sodium pump...

Mechanism of Na,K-ATPase decline during sheep red cell maturation

Energy Technology Data Exchange (ETDEWEB)

Grafova, E.; Blostein, R.

1987-05-01

Na,K-ATPase of immature and mature sheep red cells of both the high-K/sup +/ and low-K/sup +/ genotypes as well as cells of both types matured in vitro was detected using polyclonal antiserum to sheep kidney Na,K-ATPase. Following SDS-PAGE and immunoblotting, the major reactive component was the approx. 100 kDa catalytic ..cap alpha.. subunit. A less prominent band migrating as a sharper, lower molecular weight (50 kDa) component than the kidney Na,K-ATPase ..beta.. subunit is apparent in reticulocytes but not mature cells. Membranes from both genotypes showed identical immunologically reactive peptides, except for the lower intensity of the ..cap alpha.. subunit in the mature cells of the low- compared to high-K/sup +/ sheep. Following culture of both types, moderate reduction in reactivity was apparent. Immunologically reactive ..cap alpha.. subunit as well as the 50 kDa species were detected in membranous material shed into the culture medium. This material was functionally inactive (lack of both (/sup 3/H) ouabain binding and Na/sup +/-dependent phosphorylation of Na,K-ATPase). The existence in reticulocytes of an intracellular pool of ouabain binding sites is evidenced in appearance of extra sites following rapid ATP depletion and also after addition of chloroquine. Taken together, these findings are consistent with a maturation-associated decrease of sodium pumps by a process of membrane recycling, processing and, to some extent, exocytosis.

Single-molecule, structural and functional studies of Listeria monocytogenes Ca²⁺-ATPase

DEFF Research Database (Denmark)

Dyla, Mateusz

P-type ATPases constitute an essential protein family, responsible for maintaining ionic gradients across biological membranes via primary active transport (1), and alternating between the E1 and E2 states during the transport cycle, as first suggested by Post (2) and Albers (3). In the E1 state...... time. An increased Mg2+ concentration facilitated observation of transport dynamics in the wild-type-like LMCA1 mutant, arguably by inhibition of E2 dephosphorylation. LMCA1 reconstituted into proteoliposomes demonstrated ATP-dependent pumping of Ca2+ against the concentration gradient into the lumen...

Validity of Type D personality in Iceland: association with disease severity and risk markers in cardiac patients

OpenAIRE

Svansdottir, Erla; Karlsson, Hrobjartur D.; Gudnason, Thorarinn; Olason, Daniel T.; Thorgilsson, Hordur; Sigtryggsdottir, Unnur; Sijbrands, Eric J.; Pedersen, Susanne S.; Denollet, Johan

2011-01-01

textabstractType D personality has been associated with poor prognosis in cardiac patients. This study investigated the validity of the Type D construct in Iceland and its association with disease severity and health-related risk markers in cardiac patients. A sample of 1,452 cardiac patients completed the Type D scale (DS14), and a subgroup of 161 patients completed measurements for the five-factor model of personality, emotional control, anxiety, depression, stress and lifestyle factors. Th...

Critical roles of isoleucine-364 and adjacent residues in a hydrophobic gate control of phospholipid transport by the mammalian P4-ATPase ATP8A2.

Science.gov (United States)

Vestergaard, Anna L; Coleman, Jonathan A; Lemmin, Thomas; Mikkelsen, Stine A; Molday, Laurie L; Vilsen, Bente; Molday, Robert S; Dal Peraro, Matteo; Andersen, Jens Peter

2014-04-08

P4-ATPases (flippases) translocate specific phospholipids such as phosphatidylserine from the exoplasmic leaflet of the cell membrane to the cytosolic leaflet, upholding an essential membrane asymmetry. The mechanism of flipping this giant substrate has remained an enigma. We have investigated the importance of amino acid residues in transmembrane segment M4 of mammalian P4-ATPase ATP8A2 by mutagenesis. In the related ion pumps Na(+),K(+)-ATPase and Ca(2+)-ATPase, M4 moves during the enzyme cycle, carrying along the ion bound to a glutamate. In ATP8A2, the corresponding residue is an isoleucine, which recently was found mutated in patients with cerebellar ataxia, mental retardation, and dysequilibrium syndrome. Our analyses of the lipid substrate concentration dependence of the overall and partial reactions of the enzyme cycle in mutants indicate that, during the transport across the membrane, the phosphatidylserine head group passes near isoleucine-364 (I364) and that I364 is critical to the release of the transported lipid into the cytosolic leaflet. Another M4 residue, N359, is involved in recognition of the lipid substrate on the exoplasmic side. Our functional studies are supported by structural homology modeling and molecular dynamics simulations, suggesting that I364 and adjacent hydrophobic residues function as a hydrophobic gate that separates the entry and exit sites of the lipid and directs sequential formation and annihilation of water-filled cavities, thereby enabling transport of the hydrophilic phospholipid head group in a groove outlined by the transmembrane segments M1, M2, M4, and M6, with the hydrocarbon chains following passively, still in the membrane lipid phase.

V-ATPase-mediated granular acidification is regulated by the V-ATPase accessory subunit Ac45 in POMC-producing cells.

NARCIS (Netherlands)

Jansen, E.J.S.; Hafmans, T.G.M.; Martens, G.J.

2010-01-01

The vacuolar (H(+))-ATPase (V-ATPase) is an important proton pump, and multiple critical cell-biological processes depend on the proton gradient provided by the pump. Yet, the mechanism underlying the control of the V-ATPase is still elusive but has been hypothesized to involve an accessory subunit

C-terminus of the P4-ATPase ATP8A2 functions in protein folding and regulation of phospholipid flippase activity.

Science.gov (United States)

Chalat, Madhavan; Moleschi, Kody; Molday, Robert S

2017-02-01

ATP8A2 is a P4-ATPase that flips phosphatidylserine and phosphatidylethanolamine across cell membranes. This generates membrane phospholipid asymmetry, a property important in many cellular processes, including vesicle trafficking. ATP8A2 deficiency causes severe neurodegenerative diseases. We investigated the role of the C-terminus of ATP8A2 in its expression, subcellular localization, interaction with its subunit CDC50A, and function as a phosphatidylserine flippase. C-terminal deletion mutants exhibited a reduced tendency to solubilize in mild detergent and exit the endoplasmic reticulum. The solubilized protein, however, assembled with CDC50A and displayed phosphatidylserine flippase activity. Deletion of the C-terminal 33 residues resulted in reduced phosphatidylserine-dependent ATPase activity, phosphatidylserine flippase activity, and neurite extension in PC12 cells. These reduced activities were reversed with 60- and 80-residue C-terminal deletions. Unlike the yeast P4-ATPase Drs2, ATP8A2 is not regulated by phosphoinositides but undergoes phosphorylation on the serine residue within a CaMKII target motif. We propose a model in which the C-terminus of ATP8A2 consists of an autoinhibitor domain upstream of the C-terminal 33 residues and an anti-autoinhibitor domain at the extreme C-terminus. The latter blocks the inhibitory activity of the autoinhibitor domain. We conclude that the C-terminus plays an important role in the efficient folding and regulation of ATP8A2. © 2017 Chalat et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).

Diallyl tetrasulfide improves cadmium induced alterations of acetylcholinesterase, ATPases and oxidative stress in brain of rats

International Nuclear Information System (INIS)

Pari, Leelavinothan; Murugavel, Ponnusamy

2007-01-01

Cadmium (Cd) is a neurotoxic metal, which induces oxidative stress and membrane disturbances in nerve system. The garlic compound diallyl tetrasulfide (DTS) has the cytoprotective and antioxidant activity against Cd induced toxicity. The present study was carried out to investigate the efficacy of DTS in protecting the Cd induced changes in the activity of acetylcholinesterase (AChE), membrane bound enzymes, lipid peroxidation (LPO) and antioxidant status in the brain of rats. In rats exposed to Cd (3 mg/kg/day subcutaneously) for 3 weeks, a significant (P + K + -ATPase, Mg 2+ -ATPase and Ca 2+ -ATPase) were observed in brain tissue. Oral administration of DTS (40 mg/kg/day) with Cd significantly (P < 0.05) diminished the levels of LPO and protein carbonyls and significantly (P < 0.05) increased the activities of ATPases, antioxidant enzymes, GSH and TSH in brain. These results indicate that DTS attenuate the LPO and alteration of antioxidant and membrane bound enzymes in Cd exposed rats, which suggest that DTS protects the brain function from toxic effects of Cd

Inhibition of partially purified K+/H+-ATPase from guinea-pig isolated and enriched parietal cells by substituted benzimidazoles.

Science.gov (United States)

Beil, W.; Sewing, K. F.

1984-01-01

The cellular and subcellular distributions of adenosinetriphosphatases (ATPases) were examined in guinea-pig gastric mucosal cells. All cell types displayed Mg2+-ATPase and bicarbonate (HCO3-)-stimulated ATPase activity. K+-ATPase was located only in fractions derived from parietal cells. Differential and density-gradient centrifugation of material prepared from parietal cells revealed that K+-ATPase activity was located in a tubulo-vesicular membrane fraction. Enzyme activity was ten fold greater in this fraction than in a crude parietal cell homogenate. The substituted benzimidazoles, omeprazole and picoprazole, inhibited K+-ATPase (IC50 1.8 +/- 0.5 mumol l-1 and 3.1 +/- 0.4 mumol l-1, respectively). Detailed kinetic analysis indicated that these compounds were non-competitive and reversible inhibitors of the enzyme. In contrast cimetidine and verapamil were without effect on the enzyme. The relevance of the inhibition of K+-ATPase to the antisecretory activity of the benzimidazoles, in experimental animals and man, is discussed. PMID:6146367

General and specific lipid-protein interactions in Na,K-ATPase.

Science.gov (United States)

Cornelius, F; Habeck, M; Kanai, R; Toyoshima, C; Karlish, S J D

2015-09-01

The molecular activity of Na,K-ATPase and other P2 ATPases like Ca(2+)-ATPase is influenced by the lipid environment via both general (physical) and specific (chemical) interactions. Whereas the general effects of bilayer structure on membrane protein function are fairly well described and understood, the importance of the specific interactions has only been realized within the last decade due particularly to the growing field of membrane protein crystallization, which has shed new light on the molecular details of specific lipid-protein interactions. It is a remarkable observation that specific lipid-protein interactions seem to be evolutionarily conserved, and conformations of specifically bound lipids at the lipid-protein surface within the membrane are similar in crystal structures determined with different techniques and sources of the protein, despite the rather weak lipid-protein interaction energy. Studies of purified detergent-soluble recombinant αβ or αβFXYD Na,K-ATPase complexes reveal three separate functional effects of phospholipids and cholesterol with characteristic structural selectivity. The observations suggest that these three effects are exerted at separate binding sites for phophatidylserine/cholesterol (stabilizing), polyunsaturated phosphatidylethanolamine (stimulatory), and saturated PC or sphingomyelin/cholesterol (inhibitory), which may be located within three lipid-binding pockets identified in recent crystal structures of Na,K-ATPase. The findings point to a central role of direct and specific interactions of different phospholipids and cholesterol in determining both stability and molecular activity of Na,K-ATPase and possible implications for physiological regulation by membrane lipid composition. This article is part of a special issue titled "Lipid-Protein Interactions." Copyright © 2015 Elsevier B.V. All rights reserved.

Effects of short-term tocopherol (T) feeding on structure-localized protein tyrosine nitration (pTN) patterns of mitochondrial ATPase following endotoxin (LPS) challenge in beef calves.

Science.gov (United States)

Mitochondrial ATPase/Complex-V (MCV) is an electron transport chain (ETC) component needed for ATP synthesis. The ETC, exquisitely sensitive to proinflammatory mediators (PIM), generates oxynitrogen reactants leading to pTN formation as mitochondrial membrane leakage occurs. Immunohistochemical loca...

Role of Na+/K+-ATPase in Natriuretic Effect of Prolactin in a Model of Cholestasis of Pregnancy.

Science.gov (United States)

Abramicheva, P A; Balakina, T A; Bulaeva, O A; Guseva, A A; Lopina, O D; Smirnova, O V

2017-05-01

Participation of Na+/K+-ATPase in the natriuretic effect of prolactin in a cholestasis of pregnancy model was investigated. The Na+/K+-ATPase activity in rat kidney medulla, where active sodium reabsorption occurs, decreased in the model of cholestasis of pregnancy and other hyperprolactinemia types compared with intact animals. This effect was not connected with the protein level of α1- and β-subunits of Na+/K+-ATPase measured by Western blotting in the kidney medulla. Decrease in Na+/K+-ATPase activity in the kidney cortex was not significant, as well as decrease in the quantity of mRNA and proteins of the α1- and β-subunits of Na+/K+-ATPase. There were no correlations between the Na+/K+-ATPase activity and sodium clearance, although sodium clearance increased significantly in the model of cholestasis of pregnancy and other hyperprolactinemia groups under conditions of stable glomerular filtration rate measured by creatinine clearance. We conclude that the Na+/K+-ATPase is not the only mediator of the natriuretic effect of prolactin in the model of cholestasis of pregnancy.

Effects of synthetic and naturally occurring flavonoids on Na+, K+-ATPase: Aspects of the structure-activity relationship and action mechanism

International Nuclear Information System (INIS)

Hirano, T.; Oka, K.; Akiba, M.

1989-01-01

A comparative study was made of the effects of 15 synthetic and naturally occurring flavonoids on the hydrolytic activity of Na + , K + -adenosine triphosphatase (ATPase). Twelve of the flavonoids examined were mono-hydroxy or mono-methoxy derivatives. All inhibited Na + , K + -ATPase from dog kidney cortex when present at concentrations from 40-1000 μM. Flavones possessing cyclohexyl instead of the phenyl group were the most potent with IC 50 at 257-320 μM. Structure-activity relationships were observed among the following mono-substituted flavones as: (i) 2-cyclohexyl-benzopyran-4-one much-gt 2-phenyl-benzopyran-4-one; (ii) 2-cyclohexyl-7-hydroxybenzopyran-4-one > 2-cyclohexyl-6-hydroxy-benzopyran-4-one > 2-cyclohexyl-5-hydroxybenzopyran-4-one. Some flavonoids showing potent inhibitory activity were also examined for ouabain-displacement activity on human erythrocytes. Hardly and of the flavonoids were able to block [ 3 H] ouabain binding to erythrocytes. These results suggest that the mechanism by which flavonoid block Na + , K + -ATPase is not related to the cardiac glycoside-specific binding site(s) of this enzyme

Novel aspects of Na+,K+-ATPase

OpenAIRE

Aizman, Oleg

2002-01-01

Na,K-ATPase, an integral membrane protein expressed in each eukaryotic cell, serves as the major determinant of intracellular ion composition. In the current study we investigated novel aspects of Na,K-ATPase function and regulation. It is well established that Na,K-ATPase activity is regulated by reversible phosphorylation. New findings in this study are: 1) the level of intracellular Ca 2. concentration determines the functional effects of PKA and PKC-mediated Na,K-ATP...

Role of protein kinase C in regulation of Na+- and K +-dependent ATPase activity and pump function in corneal endothelial cells.

Science.gov (United States)

Hatou, Shin; Yamada, Masakazu; Mochizuki, Hiroshi; Nishida, Teruo

2009-05-01

Na+- and K+-dependent ATPase (Na,K-ATPase) plays an important role in the pump function of the corneal endothelium. We investigated the possible role of protein kinase C (PKC) in regulation of Na,K-ATPase activity and pump function in corneal endothelial cells. Confluent monolayers of mouse corneal endothelial cells were exposed to phorbol 12,13-dibutyrate (PDBu) to induce activation of PKC. ATPase activity of the cells was evaluated by using ammonium molybdate in spectrophotometric measurement of phosphate released from ATP, with Na,K-ATPase activity being defined as the portion of total ATPase activity sensitive to ouabain. Pump function of the cells was measured with a Ussing chamber, with the pump function attributable to Na,K-ATPase activity being defined as the portion of the total short-circuit current sensitive to ouabain. PDBu (10(-7) M) increased the Na,K-ATPase activity and pump function of the cultured cells. These effects of PDBu were potentiated by the cyclooxygenase inhibitor indomethacin and the cytochrome P(450) inhibitor resorufin and were blocked by okadaic acid, an inhibitor of protein phosphatases 1 and 2A. Our results suggest that PKC bidirectionally regulates Na,K-ATPase activity in mouse corneal endothelial cells: it inhibits Na,K-ATPase activity in a cyclooxygenase- and cytochrome P(450)-dependent manner, whereas it stimulates such activity by activating protein phosphatases 1 or 2A.

The non-gastric H,K-ATPase as a tool to study the ouabain-binding site in Na,K-ATPase.

NARCIS (Netherlands)

Pont, J.J.H.H.M. de; Swarts, H.G.P.; Karawajczyk, A.; Schaftenaar, G.; Willems, P.H.G.M.; Koenderink, J.B.

2009-01-01

Based on studies with chimeras between (non-)gastric H,K-ATPase and Na,K-ATPase, a model for the ouabain binding site has recently been presented (Qiu et al. J.Biol.Chem. 280 (2005) 32349). In this model, hydrogen bonds between specific amino acid residues of Na,K-ATPase and hydroxyl groups of

Structural and functional cardiac changes in myotonic dystrophy type 1: a cardiovascular magnetic resonance study

Directory of Open Access Journals (Sweden)

Hermans Mieke CE

2012-07-01

Full Text Available Abstract Background Myotonic dystrophy type 1 (MD1 is a neuromuscular disorder with potential involvement of the heart and increased risk of sudden death. Considering the importance of cardiomyopathy as a predictor of prognosis, we aimed to systematically evaluate and describe structural and functional cardiac alterations in patients with MD1. Methods Eighty MD1 patients underwent physical examination, electrocardiography (ECG, echocardiography and cardiovascular magnetic resonance (CMR. Blood samples were taken for determination of NT-proBNP plasma levels and CTG repeat length. Results Functional and structural abnormalities were detected in 35 patients (44%. Left ventricular systolic dysfunction was found in 20 cases, left ventricular dilatation in 7 patients, and left ventricular hypertrophy in 6 patients. Myocardial fibrosis was seen in 10 patients (12.5%. In general, patients had low left ventricular mass indexes. Right ventricular involvement was uncommon and only seen together with left ventricular abnormalities. Functional or structural cardiac involvement was associated with age (p = 0.04, male gender (p Conclusions CMR can be useful to detect early structural and functional myocardial abnormalities in patients with MD1. Myocardial involvement is strongly associated with conduction abnormalities, but a normal ECG does not exclude myocardial alterations. These findings lend support to the hypothesis that MD1 patients have a complex cardiac phenotype, including both myocardial and conduction system alteration.